Agradecemos a K. Gill para a assistência com a criação do mouse e os cuidados e para a gestão de laboratório. Agradeço também aos membros Mukoyama laboratório para assistência técnica. O financiamento foi fornecido pelo Programa de Pesquisa Intramural dos Institutos de Saúde Naitonal.

1. Coleta de rato Pele Limb embrionárias (E13.5 E17.5 ~) <ol><li> Euthanize conectado fêmeas através de um procedimento aprovado. De acordo com nosso protocolo aprovado animais, as fêmeas são sacrificados por exposição ao CO 2 e, em seguida, assegurada por deslocamento cervical. Lay o animal à sua toalha de papel absorvente e deixe-o completamente em 70% EtOH / H 2 O de uma garrafa squeeze. </li><li> Dissecar o útero intacto e coloque-o em um prato 100 15 mm x Petri contendo solução gelada de Hanks salina balanceada (HBSS) para lavar o sangue. </li><li> Separado e dissecar o embrião. Remover o âmnio muito fina a partir do embrião. </li><li> (Opção) Dissecar um único embrião em um 35 10 mm x placa de Petri se cada embrião deve ser genotipados. Dissecados cauda é transferido para um tubo de 0,2 ml PCR para genotipagem. </li><li> Cortar as patas dianteiras do embrião e transferência forelimbs por uma pinça de anel em 24 placa bem contendo 2 ml de gelado paraformaldeído frescos 4% (PFA) em tampão fosfato salino (PBS). </li><li> Corrigir o forelimbs com suave mistura na Mixer Nutator a 4 ° C durante a noite. </li><li> No dia seguinte, retire a PFA e lavar três vezes por 5 min em 2 ml de PBS com leve mistura no Mixer Nutator à temperatura ambiente. </li><li> Transferir os membros anteriores em metanol 100% (MeOH) e armazená-los freezer a -20 ° C enzima (o freezer com controle de temperatura crítica e sem função de descongelação automática). Anticorpos primárias constantes da Tabela 1 trabalho após o tratamento MeOH 100%. </li><li> Peeling off do membro anterior, usando uma pinça fina. Pele dos membros, quando desidratado, deve separar facilmente a partir do membro. Primeiro lugar o membro com o lado ventral (lado da palma) voltado para cima. Em seguida, usando uma pinça fina, cortar a pele, como mostrado no vídeo. Próxima dissecar todo o membro inteiro, descascando a pele suavemente, sem qualquer dano. </li></ol> 2. Toda montagem de coloração imuno-histoquímica de Skins Limb <ol><li> Hidratar as peles de membros em 5ml de polipropileno tubos de fundo redondo, incubando através da série graduada de MeOH / PBT (PBS + 0,2% Triton X-100) (75%, 50%, 25%) por 5 min cada. Note-se que a troca de soluções deve ser cuidadoso para não danificar as peles dos membros. </li><li> Lavar duas vezes por 5 min em PBT com suave mistura na Mixer Nutator à temperatura ambiente. </li><li> Bloquear o membro com peles de cabra ou 10% de soro / PBS 0,2% TX100 tampão de bloqueio por anticorpos de cabra secundário ou 10% Donkey soro / PBS 0,2% TX100 tampão de bloqueio por anticorpos burro secundário por 2 horas com a mistura suave no Mixer Nutator em temperatura ambiente. </li><li> Coloque as cascas dos membros em um prato de 35 10 mm x Petri e transferência por uma pinça anel em microcentrífuga-2ml tubo com 800μl de anticorpos primários (diluição adequada, conforme listado nas tabelas) no tampão de bloqueio (ou 10% de soro de cabra / PBS 0,2 TX100% ou 10% Donkey soro / PBS 0,2% TX100). Incubar as peles membro com suave mistura na Mixer Nutator a 4 ° C durante a noite. Note-se que vários anticorpos primários derivados de espécies diferentes (por exemplo, anticorpo monoclonal de rato + anticorpo policlonal de coelho) podem ser usados ​​simultaneamente. </li><li> No dia seguinte, coloque as peles do membro em um prato de 35 10 mm x Petri e transferência por uma pinça anel em polipropileno 5ml de fundo redondo de tubo com 4ml da solução de lavagem (ou 2% de soro de cabra / PBS 0,2% ou 2 TX100 Donkey% de soro / PBS 0,2% TX100). </li><li> Lavar cinco vezes por 15 minutos com a mistura suave no Mixer Nutator à temperatura ambiente. </li><li> Coloque as cascas dos membros em um prato de 35 10 mm x Petri e transferência por uma pinça anel em microcentrífuga-2ml tubo com 800μl de anticorpos secundária no tampão de bloqueio (ou 10% de soro de cabra / PBS 0,2% ou 10% TX100 Donkey soro / PBS 0,2% TX100). Normalmente usamos anticorpos secundários a partir de Jackson ImmunoResearch (Cy3, Cy5, 1:300 diluições) ou Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 diluição). Filtrar a solução usando anticorpo secundário 0.22μm PVDF filtros de membrana seringa para remover as partículas agregadas dos anticorpos secundários. Incubar as peles dos membros no escuro ou envolto com papel alumínio por 1 hora com a mistura suave na Mixer Nutator à temperatura ambiente. Note-se que diferentes fluorescentes conjugados anticorpos secundários derivados de espécies diferentes podem ser usados ​​simultaneamente. </li><li> Coloque as cascas dos membros em um prato de 35 10 mm x Petri e transferência por uma pinça anel em 5ml de polipropileno tubos de fundo redondo com 4ml da solução de lavagem (ou 2% de soro de cabra / PBS 0,2% ou 2% TX100 Donkey soro / PBS 0,2% TX100). Lavar cinco vezes por 15 min no escuro ou envolto com folha de alumínio com a mistura suave no Mixer Nutator à temperatura ambiente. </li><li> (Opção para contracoloração contra núcleo) Incubar as peles membro com 4ml da solução de lavagem (ou cabra 2% de soro / PBS 0,2% ou 2% TX100 Donkey soro / PBS 0,2% TX100) com To-Pro-3 (Invitrogen T3605 , 1:3000 diluição) no escuro ou envolto com papel alumínio por 10 minutos com a mistura suave no Mixer Nutator na sala de temperature. Então, lave três vezes por 5 min com 4ml da solução de lavagem (ou 2% de soro de cabra / PBS 0,2% ou 2% TX100 Donkey soro / PBS 0,2% TX100) no escuro ou envolto por folhas de alumínio com a mistura suave no o Mixer Nutator à temperatura ambiente. Note-se que a coloração forte e específica para núcleos é observado para To-pro-3 em uma emissão específica (HeNe excitação nm 633). </li></ol> 3. Montagem do Skins Limb no slide <ol><li> Coloque as cascas em um membro 35 10 mm x placa de Petri. Remover poeiras, cristais, fibras da camada interna da pele, usando uma pinça fina sob o estereomicroscópio com iluminação baixa para evitar a foto branqueamento extensivo. </li><li> Transferir os skins membro para lâmina de microscópio adesiva por uma pinça de anel. Coloque as peles com a camada interna deitada para cima no slide (ou seja, para lamela). Achate as peles cuidadosamente usando uma pinça fina e remover carry-over tampão de lavagem por Kimwipe. </li><li> Montagem em anti-fade meios de montagem sem bolhas de ar. Nós usamos 25 &quot;x25&quot; lamela. Cura em uma superfície plana no escuro (por exemplo, as amostras montado usando Prolongar reagente de ouro são colocadas durante a noite no escuro à temperatura ambiente antes de visualizar). Para armazenamento a longo prazo, vedar a lamela para o slide e armazenar a 4 ° C. </li></ol> 4. Microscopia confocal <ol><li> Criar lasers apropriados para fluoróforos. Usamos Leica TCS microscópio confocal SP5 com três fontes de laser de argônio, incluindo 488nm (para Alexa Fluor 488 e GFP), 561nm DPSS (por Alexa Fluor 568 e Cy3) e 633 nm HeNe (por Alexa Fluor 633, Cy5 e Para-Pro-3) . </li><li> Use a ferramenta de busca sequencial para evitar ou reduzir a interferência em que todos os corantes em amostras de dobrar ou triplicar manchada será animado ao mesmo tempo. No modo de varredura seqüencial, as imagens serão gravadas em uma ordem seqüencial. </li><li> Mais informações gerais sobre corantes fluorescentes e lasers para a excitação pode ser fundada em &quot;Microscopia Confocal para biólogos&quot; por Hibbs (2004) </li></ol> 5. Resultados representante Todo-mount microscopia confocal triple-label immnofluorescence em pele de rato forelimb em E15.5 com anticorpos para o marcador de células pan-endotelial PECAM-1 (Figura 1A-C, D azul), o marcador neurofilamento 2H3 (Figura 1A verde), e o marcador de células musculares lisas αSMA (Figura 1A, B vermelho) revelou um padrão característico de ramificação das artérias αSMA +, alinhado com 2H3 + nervos periféricos na pele dos membros. Além de ramificação dos vasos sanguíneos, este membro modelo vascularização da pele é usada para estudar padrões de ramificação de vasos linfáticos utilizando anticorpos para o marcador de células endoteliais linfáticas lyve-1 (Figura 1D, E vermelha) e Neuropilin2 (NRP2) (Figura 1D, F verde ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Figura 1" /> Figura 1. (AC) Artérias alinhar com os nervos periféricos na pele dos membros embrionárias. Todo-mount-label triple microscopia confocal immnofluorescence com anticorpos para o marcador de células pan-endotelial PECAM-1 (AC, azul), o marcador neurofilamento 2H3 (A, verde), eo marcador de células musculares lisas αSMA (A e B, vermelho ) é mostrado. No E15.5, 2H3 + nervos periféricos (setas abertas) associado com artérias (setas), que são cobertos por αSMA + células musculares lisas. (DF) vasculatura linfática na pele dos membros embrionárias. Label triple-microscopia confocal immnofluorescence com anticorpos para o marcador de células pan-endotelial PECAM-1 (D, azul), o marcador de células endoteliais linfáticas lyve-1 (D e E, vermelho) e Neuropilin2 (NRP2) (D e F, verde ) é mostrado. Vasos linfáticos são visualizados por ambos lyve-1 e NRP2, enquanto lyve-1 também é expressa por um subconjunto de macrófagos (setas abertas). Barra de escala: 100μm.

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

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O sistema vascular é fundamental para o desenvolvimento do órgão durante a embriogênese, bem como para a manutenção de órgãos e funções reprodutivas nos adultos, pois fornece oxigênio e nutrientes suficientes para os órgãos. Rede vascular adequada é bem estabelecido com processos complexos e multi-passo a angiogênese em que o pré-existentes da rede capilar é reorganizada com estruturas altamente ramificada e hierarquizada. Apesar de inúmeros trabalhos têm demonstrado que uma variedade de moléculas es...

Anticorpos Pan-endotelial marcador de células <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> PECAM-1 </td><td> Hamster Armenian (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> Diluição 1:100 # 1 </td></tr><tr><td> PECAM-1 </td><td> Rat (M) </td><td> BD </td><td> 553369 </td><td> 1:300 diluição </td></tr><tr><td> VEGFR2 </td><td> Rat (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> Diluição de 1:200 </td></tr><tr><td> CD34 </td><td> Rat (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 diluição </td></tr><tr><td> Colágeno IV </td><td> Coelho (P) </td><td> AbD Serotec </td><td> 2150-1470 </td><td> 1:300 diluição # 2 </td></tr></tbody></table> Marcador de células endoteliais arteriais <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> Neuropilin1 </td><td> Coelho (P) </td><td> O laboratório Kolodkin Alex # 3 </td><td></td><td> 1:3000 diluição </td></tr><tr><td> Unc5H2 </td><td> Bode (P) </td><td> R &amp; D </td><td> AF1006 </td><td> Diluição de 1:200 </td></tr></tbody></table> Venosa marcador de células endoteliais <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> EphB4 </td><td> Bode (P) </td><td> R &amp; D </td><td> AF446 </td><td> Diluição 1:100 </td></tr></tbody></table> Linfática marcador de células endoteliais <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> Lyve-1 # 4 </td><td> Coelho (P) </td><td> Abcam </td><td> ab14917 </td><td> Diluição de 1:200 </td></tr><tr><td> Lyve-1 # 4 </td><td> Rat (M) </td><td> MBL </td><td> D225-3 </td><td> Diluição de 1:200 </td></tr><tr><td> Prox-1 </td><td> Coelho (P) </td><td> Chemicon </td><td> AB5475 </td><td> 1:1000 diluição </td></tr><tr><td> Prox-1 </td><td> Bode (P) </td><td> R &amp; D </td><td> AF2727 </td><td> 01:50 diluição </td></tr><tr><td> Neuropilin2 </td><td> Coelho (P) </td><td> Sinalização celular </td><td> 3366 </td><td> 1, 100 de diluição </td></tr><tr><td> Podoplanin </td><td> Hamster sírio (M) </td><td> Hibridoma Banco </td><td> 8.1.1 </td><td> Diluição de 1:200 </td></tr></tbody></table> Músculo liso marcador de células / pericyte <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> αSMA-Cy3 </td><td> Mouse (M) # 5 </td><td> Sigma </td><td> c-6198 </td><td> 1:500 diluição # 6 </td></tr><tr><td> NG2 </td><td> Coelho (P) </td><td> Chemicon </td><td> AB5320 </td><td> Diluição de 1:200 </td></tr><tr><td> SM22α </td><td> Coelho (P) </td><td> Abcam </td><td> ab14106 </td><td> Diluição de 1:200 </td></tr></tbody></table> Anticorpos para repórter GFP <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> GFP </td><td> Coelho (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 diluição </td></tr><tr><td> GFP </td><td> Rat (M) </td><td> Nacalai tesque </td><td> 04404-84 </td><td> 1:1000 diluição </td></tr><tr><td> GFP </td><td> Chick (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 diluição </td></tr></tbody></table> Anticorpos para repórter LacZ <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> β-gal </td><td> Coelho (P) </td><td> MP Biomédica </td><td> 55976 </td><td> 1:5000 diluição </td></tr><tr><td> β-gal </td><td> Bode (P) </td><td> AbD Serotec </td><td> 4600-1409 </td><td> 1:500 diluição </td></tr><tr><td> β-gal </td><td> Chick (P) </td><td> Abcam </td><td> ab9361 </td><td> Diluição de 1:200 </td></tr></tbody></table> Anticorpos para axônio periférico <table border="1"><tbody><tr><td> Anticorpo </td><td> Espécies </td><td> Companhia </td><td> Catálogo # </td><td> Condição de trabalho </td></tr><tr><td> 2H3 </td><td> Mouse (M) # 7 </td><td> Hibridoma Banco </td><td> 2H3 </td><td> Diluição de 1:200 </td></tr><tr><td> Tuj1 </td><td> Mouse (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> 1:500 diluição </td></tr><tr><td> Periferina </td><td> Coelho (P) </td><td> Chemicon </td><td> AB1530 </td><td> 1:1000 diluição </td></tr></tbody></table> Anticorpos para as células de Schwann migrando <table border="1"><tbody><tr><td> BFABP </td><td> Coelho (P) </td><td> O laboratório de Thomas Müller # 9 </td><td></td><td> 1:3000 diluição </td></tr></tbody></table> (P): anticorpo policlonal, (M): anticorpo monoclonal # 1: Goat anti-armênio hamster-Cy3 anticorpos (Jackson ImmunoResearch 127-165-160) deve ser usado como um anticorpo secundário. # 2: O Colágeno IV anticorpos podem ser usados ​​para detectar os vasos sanguíneos após a hibridização in situ. # 3: O anticorpo Neuropilin1 é gentilmente cedidas pelo laboratório do Kolodkin Alex na Johns Hopkins University. Ovelhas anti-anticorpo humano Neuropilin1 está disponível em P &amp; D (AF3870), embora não tenha testado ainda. # 4: Os anticorpos lyve-1 também detectar um subconjunto de macrófagos na pele embrionárias. # 5: O anticorpo anti-rato αSMA é anticorpo monoclonal IgG2a. # 6: O Cy3-conjugados de anticorpos αSMA é incubada por 1 hora à temperatura ambiente, juntamente com secanticorpos secundária para outros anticorpos primários. # 7: 2H3 anticorpo é rato anticorpo IgG1 monoclonal contra neurofilamento. # 8: Tuj1 anticorpo é rato de anticorpos monoclonais contra IgG2a Neuron-specific classe III beta-tubulina. # 9: O BFABP anticorpos (cérebro-específica de ácidos graxos binding protein) é gentilmente cedidas pelo laboratório do Thomas Müller em Max-Delbrück-Center de Medicina Molecular.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Todo-mount análise imunohistoquímica para a imagem latente toda a vasculatura é fundamental para a compreensão dos mecanismos celulares de ramificação morfogênese. Nós desenvolvemos o membro modelo vascularização da pele para estudar o desenvolvimento vascular em que um pré-existentes primitiva do plexo capilar é reorganizado em uma rede hierarquicamente ramificada vascular. Todo-mount microscopia confocal com rotulagem múltiplos permite imagem robusta dos vasos sanguíneos intacta, assim como seus componentes celulares, incluindo células endoteliais, pericitos e células musculares lisas, usando marcadores fluorescentes específicos. Avanços neste modelo vasculatura membro pele com estudos genéticos têm melhorado a compreensão dos mecanismos moleculares do desenvolvimento vascular e padronização. O membro modelo vasculatura da pele tem sido usada para estudar como os nervos periféricos fornecer um modelo espacial para a diferenciação e padronização das artérias. Este artigo descreve um protocolo de vídeo simples e robusto para corar vasos sanguíneos vascular intacto com anticorpos específicos e fluorescentes anticorpos secundários, que é aplicável para os órgãos vascularizados embrionárias, onde somos capazes de acompanhar o processo de desenvolvimento vascular.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Nós introduzimos um conjunto de montagem imunohistoquímica e microscopia confocal de varredura a laser com vários rotulagem para analisar a formação de uma intrincada rede vascular em pele de rato membro embrionárias.

Todo-mount Análise imuno-histoquímica para Vascularização embrionárias Limb pele: um sistema modelo para estudar Morfogênese Branching Vascular em Embrião

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K visualizações.  National Heart, Lung, and Blood Institute, National Institutes of Health. Nós introduzimos um conjunto de montagem imunohistoquímica e microscopia confocal de varredura a laser com vários rotulagem para analisar a formação de uma intrincada rede vascular em pele de rato membro embrionárias.

Vídeo: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte Whole dupla hibridização in situ de embriões de galinha precoce

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biologia

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para coloração Whole Mount anticorpos em Pintainho

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura do mouse pulmão embrionário, um sistema para avaliar os mecanismos moleculares de Ramificação

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; Em situ</em&gt; Hibridação em embriões de Mount inteiros e histologia de células Adaptado para Elasmobrânquios Marinhos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Rato Desenvolvimento Embrionário em todo um sistema de cultura de embriões sem soro

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análise de apoptose em embriões Zebrafish por Whole-mount imunofluorescência para detectar Caspase 3 ativado

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...