Os autores não têm reconhecimento.

Todos os procedimentos experimentais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade Metropolitana de Tóquio (IACUC). Os animais foram alojados e manuseados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório e o Guia para o Cuidado e Uso de Animais Experimentais do Conselho Canadense de Cuidados com Animais. Utilizamos animais criados e mantidos no Laboratório de Morfologia Funcional, Departamento de Ciências da Saúde da Fronteira, Universidade Metropolitana de Tóquio, Japão. 1. Cuidados com animais e habitação <ol><li> Obtenha 7 S. murinus (3 fêmeas e 4 machos pesando 70-90 g) de uma colônia de criação fechada. </li><li> Cale o S. murinus individualmente após o desmame (20 d após o nascimento ) em gaiolas de plástico equipadas com uma caixa de ninho de madeira contendo tiras de papel. Mantenha-os em uma sala de animais convencionalmente condicionada: 23-27 ° C, sem controle de umidade e uma luz de 12:12 h: ciclo escuro. Supply commercPellets de trutas e água ad libitum. </li></ol> 2. Preparação de Tecidos <ol><li> Para preparar 4% (p / v) de paraformaldeído (PFA), dissolver 40 g de PFA em 1000 mL de solução salina tamponada com fosfato 0,01 M (PBS, pH 7,4). Em uma aspiradora, misture usando um vórtice até a solução ficar limpa. Armazene 4% de PFA O / N a 4 ° C. Cuidado: Use equipamento de proteção pessoal apropriado (PPE) ao manusear o PFA. </li><li> Para perfurar e consertar o animal, anestesiar com éter e depois dar-lhe uma injeção intraperitoneal de somnopentilo (pentobarbital sódico, 0,6 mL / kg de peso corporal). </li><li> Depois que o animal é completamente narcotizado, abra a cavidade abdominal com um bisturi, criando uma incisão abdominal na linha média com 3 cm de comprimento. Ao incitar a veia cava inferior para exsanguinating, insira um catéter retrogradável na aorta abdominal no nível imediatamente acima da bifurcação desta artéria nas artérias ilíacas comuns. </li><li> Injetar somnopentil (pentobarbital assimDium, 1,0 mL / kg de peso corporal). Após a eutanásia completa, perfusar com PBS 0,01 M (pH 7,4) e depois com 4% de PFA numa aspiradora. </li><li> Após a perfusão, injete 2-3 mL de látex branco de neoprene (diluição do látex de neoprene branco 3: 2 com água destilada (DW)) para rotular os vasos sanguíneos. </li><li> Extraia os órgãos abdominais, incluindo o fígado, a vesícula biliar, o ducto biliar comum, o duodeno e o pâncreas em bloco, com os nervos e vasos relacionados. </li><li> Injetar aproximadamente 0,1 mL de látex azul de neoprene (adicionar uma gota de tinta azul a 10 mL do látex de neoprene branco diluído) até a vesícula biliar para rotular o sistema biliar. </li><li> Pós-fixação durante a noite com 4% de PFA a 4 ° C para imunomarcação total. Atenção: o PFA é tóxico. Evite manipular diretamente o PFA. O PFA deve ser usado em uma chaminé. </li></ol> 3. Imunohistoquímica de montagem total Nota: Ao longo do protocolo, incluindo durante a lavagem, incubação de anticorpos e coloração,O espécime deve permanecer no nutator, balançando suavemente à TA ou a 4 ° C. <ol><li> Dia 1: Tratamento enzimático e inativação da potencial peroxidase endógena <ol><li> Lave o espécime preparado com DW 4x por 1 h cada à temperatura ambiente em um frasco de vidro de tamanho apropriado. Balance o espécime gentilmente sobre o nutator para remover o PFA. Evite danificar o espécime ao trocar soluções. </li><li> Para preparar 1% (p / v) de ácido ortoperiodico, dissolva 1 g de ácido ortoperiodico em 100 mL de DW. </li><li> Incubar a amostra em 1% de ácido ortoperiodico durante 20 minutos à TA para evitar qualquer reação intrínseca à peroxidase. </li><li> Prepare 0,004% (p / v) de papaína por dissolução de 0,004 g de papaína em 100 mL de tampão Tris-HCl 0,025 mol / L (pH 7,6). </li><li> Lave o espécime com DW por 10 minutos à TA. </li><li> Incubar o espécime em 0,004% de papaia recém-preparada durante 2 h a 37 ° C em banho a temperatura constante com balanço suave. </li><li> Lave o espécime com DW por 50-60 minutos na RT. </li><li> Armazene o espécime em 4% de PFA a 4 ° CO / N. </li></ol></li><li> Dia 2: tratamento enzimático <ol><li> Lave o espécime armazenado com DW 4 vezes durante 1 h cada à TA. </li><li> Incubar o espécime em 0,004% de papaia recém-preparada durante 2 h a 37 ° C em banho a temperatura constante com balanço suave. </li><li> Lave o espécime com DW durante 50-60 minutos à RT. Armazene o espécime em 4% de PFA a 4 ° CO / N. </li></ol></li><li> Dia 3: tratamento de congelação <ol><li> Lave o espécime armazenado com DW 4x durante 1 h cada à temperatura ambiente, como acima. </li><li> Preparar 2,5% (p / v), 5% (p / v) e 10% (p / v) de sacarose dissolvendo 2,5 g, 5 g e 10 g de sacarose, respectivamente, em 100 mL de PBS 0,01 M ( PH 7,4). </li><li> Mergulhe o espécime em 2,5% (p / v), 5% (p / v) e 10% (p / v) de sacarose por 30 min cada. </li><li> Congele o espécime a -20 ° C durante 30 a 60 min e depois descongelá-lo completamente à TA; Repita o ciclo 3x. </li><li> TopoRepare 2% de Triton X-100 (v / v), adicione 2 mL de Triton X-100 a 100 mL de PBS 0,01 M (pH 7,4). </li><li> Armazene o espécime em 2% de Triton X-100 a 4 ° CO / N. </li></ol></li><li> Dia 4: incubação primária de anticorpos <ol><li> Prepare a solução de diluição do anticorpo primário dissolvendo 0,2 g de albumina de soro bovino (BSA), 0,3 mL de Triton X-100 e 0,1 g de azida de sódio em 100 mL de PBS 0,01 M (pH 7,4). Atenção: A azida de sódio é extremamente tóxica. A inalação e o toque devem ser evitados. Use PPE apropriado. </li><li> Diluir o anticorpo primário (NFP) 1: 600 no tampão de diluição acima (passo 3.4.1). Incube o espécime com anticorpo NFP a 4 ° C por 3-6 dias, mantendo o espécime balançando suavemente no nutator. Espécimes maiores exigirão tempos de incubação aumentados. </li></ol></li><li> Incubação secundária de anticorpos <ol><li> Lave o espécime em PBS 4x durante 1 h cada à TA para remover o anticorpo primário não ligado. </lI&gt; <li> Preparar a solução de diluição para o anticorpo secundário dissolvendo 0,2 g de BSA e 0,3 mL de Triton X-100 em 100 mL de PBS 0,01 M (pH 7,4). </li><li> Diluir o anticorpo secundário (IgG-HRP anti-rato anti-rato purificado por afinidade conjugado com peroxidase) 1: 600 no tampão de diluição (passo 3.5.2). Incube o espécime com o anticorpo secundário a 4 ° C durante 3 dias e mantenha o espécime balançando suavemente na porca. </li></ol></li><li> Coloração <ol><li> Prepare a solução de coloração DAB adicionando 0,002 g de tetrahidrocloreto de 3,3&#39;-diaminobenzidina (DAB) a 100 mL de tampão Tris-HCl 0,05 mol / L (pH 7,6) sob uma aspiradora, mantendo a solução no escuro. </li><li> Lave os espécimes à RT em PBS 4x por 1 h cada. </li><li> Adicione 10 μL de 30% de H 2 O 2 em solução de coloração DAB recém-preparada e incuba com a solução a 4 ° CO / N ou durante 3 dias enquanto se meca suavemente no nutator. </li><li> Pare a reação por plaCing o espécime em PBS com 0,04% de azida de sódio quando a intensidade de coloração ideal é atingida. NOTA: Evite o uso de azida de sódio até este passo, pois inibe a peroxidase de rábano. </li></ol></li><li> Imaginando o tecido manchado <ol><li> Transfira cuidadosamente o espécime para um prato de vidro petri (5 cm de altura, 10 cm de diâmetro) contendo PBS. Capture imagens de montagem total usando uma câmera montada em um estereomicroscópio. NOTA: O tecido manchado de montagem total deve ser fotografado enquanto mergulhou completamente em PBS em um prato de vidro transparente. </li></ol></li></ol>

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Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+neurons+provide+nicotinic+fast+synaptic+input+to+sphincter+of+Oddi+neurons+in+guinea+pig.">Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig.</a> Am. J. Physiol. 277, G226-G234 (1999).</li><li>Seo, J. H., Cho, S. S., Lee, I. S., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+neuropeptidergic+properties+of+the+duodenal+neurons+projecting+to+the+gallbladder+in+the+golden+hamster.">Anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster.</a> Arch. Histol. Cytol. 65 (4), 317-321 (2002).</li><li>Padbury, R. T., Furness, J. B., Baker, R. A., Toouli, J., Messenger, J. 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Os autores não declaram conflitos de interesse.

Este trabalho descreve o procedimento experimental para a visualização da inervação do trato biliar extra-hepático usando um anticorpo contra a proteína do neurofilamento. Este protocolo foi adaptado do protocolo descrito por Kuratani e Tanaka 1 , 2 , 3 . Para esta experiência, planeje a linha de tempo para cada uma das experiências, já que a abordagem de coloração total continua por 2-3 semanas. O recipiente que contém o tecido deve ser rodado em um nutator em todos os momentos, exceto durante o processo de congelamento / descongelamento. Particularmente durante a incubação com os anticorpos primário e secundário, a amostra foi colocada em uma câmara de armazenamento fria enquanto girava em uma porca para garantir a exposição uniforme da amostra às soluções de reação. Várias soluções - 1% (p / v) de ácido ortoperiodico, 0,004% (p / v) de papaina e o tampão de diluição para o antioxidante primário / secundárioMorre - deve ser feito fresco para cada experimento. Ao longo do protocolo, para evitar tocar e danificar o espécime ao mudar as soluções, as soluções devem ser derramadas em vez de serem removidas com fórceps 15 . Após a fixação com PFA, as amostras devem ser suficientemente lavadas com DW ou PBS. Além disso, o tratamento enzimático com a incubação de papaína é importante. O calor e a enzima utilizados para recuperação de antígenos ajudam a preparar o tecido para a penetração de anticorpos. Para aumentar a penetração das soluções, a membrana externa do tecido deve ser interrompida por um tratamento de congelação a -20 ° C durante 30 min ou -80 ° CO / N 15 . É importante submergir completamente a amostra experimental em volumes adequados de solução tampão para garantir que a solução possa incubar todo o espécime. O tempo de incubação do anticorpo primário deve ser empiricamente determinadoD para diferentes tecidos e tamanhos de amostra. Normalmente, deve ser de 3 dias para uma amostra de 2-3 cm. A diluição ideal deve ser determinada empiricamente para cada anticorpo. Uma diluição a 1: 600 é recomendada para os anticorpos primário e secundário quando se utiliza o tampão de anticorpo apropriado. Este trabalho descreve, em detalhes, o protocolo de uma abordagem imuno-histoquimica de montagem completa versátil para revelar a expressão da proteína neurofilamentar no trato biliar de S. murinus . Este método pode ser usado para analisar a inervação de todos os órgãos viscerais em muitas espécies. Além disso, este protocolo também pode ser adaptado para testar outros anticorpos neuronais, mas a otimização dos anticorpos deve ser realizada. No entanto, a técnica foi limitada na rotulagem do tecido nervoso raso e na construção de um modelo tridimensional de inervação. Além disso, não conseguiu identificar as características das fibras nervosas ( ou seja, simpatizante ou parSimpático, motor ou sensorial, etc. ).

A muscaria Musk Muscus , Suncus murinus , pertence à ordem Insectivora e Soricidae familiar. Este pequeno mamífero é distribuído em todo o Sudeste Asiático e habita casas e áreas gramadas que se situam perto de habitações humanas ou canetas de gado 4 . Esta espécie exibe características morfológicas gerais mais semelhantes aos humanos do que outros animais de laboratório, como o mouse, o rato e o coelho 5 . Anteriormente, foi utilizada imunomarquência total com um marcador de neurônio periférico para a proteína de neurofilamento de S. murinus (NFP) para rotular os nervos periféricos no pâncreas 6 , a papila duodenal principal 7 , o píloro 8 , a vesícula biliar 5 e o trato biliar extra-hepático 9 . NFP é um complexo macromolecular que faz parte do citoesqueleto neuronal maduro. Proteína relacionada ao neurofilamentoMede as interações entre NFP e o zygosoma. Tanto a função enzimática quanto a estrutura das proteínas de ligação são reguladas pelo NFP 10 . O complexo NFP é composto de três polipéptidos: NF-L (70 kDa), NF-M (150-160 kDa) e NF-H (200 kDa). O NFP pode ser encontrado em axônios neuronais no sistema nervoso central e periférico. O anticorpo anti-NFP humano mostrou rotular os axônios do sistema nervoso central e periférico. Investigações anatômicas e eletrofisiológicas demonstraram que o esfíncter, a vesícula biliar e o trato gastrointestinal proximal estão conectados 11 , 12 , 13 , 14 . No entanto, as características morfológicas de suas conexões neuronais ainda não foram definidas. Este trabalho demonstra o uso de um método de coloração imuno-histoquimica de todo o suporte para rotular a inervação do S. murinus bilCom um anticorpo NFP.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
			<td style="width:119px;">312-33</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

Este trabalho descreve detalhadamente o método de coloração de imuno-histoquímica total, usando anticorpo de proteína de neurofilamento para rotular a inervação do trato biliar em Suncus murinus ( S. murinus &#160; ). Primeiro, a amostra foi dissecada de S. murinus e fixada em paraformaldeído a 4% (PFA). Em segundo lugar, foram realizados um tratamento enzimático e potencial inativação da peroxidase endógena. O espécime foi então exposto ao anticorpo primário, anticorpo anti-proteína de neurofilamento, durante 3-6 dias. Foi então incubado com o anticorpo secundário conjugado com peroxidase de rábano. A reação de cor foi revelada fazendo reagir o espécime com um substrato de 3,3&#39;-diaminobenzidina (DAB). Este método pode ser aplicado para analisar a inervação de todos os órgãos viscerais. Além disso, este protocolo também pode ser adaptado para testar outros anticorpos neuronais, mas a otimização do antibodiO primeiro deve ser feito primeiro. Este método foi originalmente introduzido por Kuratani e Tanaka 1 , 2 , 3 .

As Figuras 1 a 4 mostram resultados típicos para fibras nervosas NFP positivas no trato biliar extra-hepático em S. murinus . O anticorpo contra NFP reproduzivelmente rotulou a inervação em toda a imagem do trato extra-hepático biliar ( Figura 1 ), vesícula biliar ( Figura 2 ), ducto biliar superior ( Figura 3 ) e área de papila duodenal ( Figura 4 ), com alta especificidade e mínimo fundo. Para todos os tecidos do espécime, independentemente da forma e tamanho, os nervos tegmentais podem ser tingidos ao mesmo tempo. Além da inervação do tracto biliar extra-hepático, a densidade de corrida e distribuição do vago do esôfago e do estômago foi exibida inequivocamente ( Figura 1 ). <p clAss = &quot;jove_content&quot; fo: keep-together.within-page = &quot;1&quot;&gt; À medida que os vasos sanguíneos da vesícula biliar foram marcados com látex branco de neoprene, fibroses nervosas finas foram claramente demonstradas com alto contraste. A densidade de inervação foi maior no pescoço do que no fundo da vesícula biliar ( Figura 2 ). No ducto biliar superior, dois tipos de feixes nervosos foram rotulados. Os feixes de nervos finos formaram uma rede irregular e densa de nervos, correu de forma adesiva e residia no trato biliar; Os feixes neurais mais espessos foram distribuídos paralelamente à superfície do trato biliar ( Figura 3 ). O ducto biliar comum foi marcado com látex azul de neoprene e os vasos com látex branco de neoprene. As fibras nervosas finas no final do ducto biliar comum e a área de papila duodenal foram claramente demonstradas com alta c<html> Ontrast ( Figura 4 ). <img alt="figura 1" class="xfigimg" src="/files/ftp_upload/55483/55483fig1.jpg" /> Figura 1: Fibras Nervais NFP-positivas no Trato Biliar, Esófago e Estômago. As setas mostram o vago que corre ao longo do esôfago até o estômago. CBD, Common Bile Duct; Es, esôfago; St, estômago. Barra de escala = 1.300 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" class="xfigimg" src="/files/ftp_upload/55483/55483fig2.jpg" /> Figura 2: Fibras nervosas positivas para NFP na vesícula biliar. Os vasos sanguíneos da vesícula biliar foram rotulados com látex branco. Inervação abundante ocorreu no pescoço da vesícula biliar (flechas). Barra de escala = 1000 μm.Ref = &quot;https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig2large.jpg&quot; target = &quot;_ blank&quot;&gt; Clique aqui para ver uma versão maior desta figura. <img alt="Figura 3" class="xfigimg" src="/files/ftp_upload/55483/55483fig3.jpg" /> Figura 3: Fibras nervosas NFP positivas no tubo biliar superior. Foram observados dois tipos de feixes nervosos. Um tipo era os feixes de nervos finos que funcionavam de forma adesiva e residiam no trato extra-hepático biliar (setas); O outro tipo foi feixes neurais mais espessos que foram distribuídos paralelamente à superfície do trato biliar extra-hepático e correu entre a vesícula biliar e o duodeno (triângulos). PV, Portal Vein. Barra de escala = 650 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" class= &quot;Xfigimg&quot; src = &quot;/ files / ftp_upload / 55483 / 55483fig4.jpg&quot; /&gt; Figura 4: Fibras nervosas positivas para NFP na área de papila Duodenal. O ducto biliar comum foi rotulado com látex azul e os vasos com látex branco (*). As setas mostram a inervação do fim do ducto biliar comum, e os triângulos mostram a inervação da área da papila duodenal (P), que vem da via biliar comum e dos vasos. Barra de escala = 1000 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55483/55483fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Uma abordagem imuno-histoquímica de montagem total, para visualizar a expressão da proteína de neurofilamento no trato biliar extra-hepático em Suncus murinus. É apresentado aqui. Este protocolo pode ser usado para analisar a inervação de todos os órgãos viscerais em S. murinus ou outras espécies.

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

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Nanyang Technological University

Research

JoVE Journal

Biology

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K visualizações.  Tokyo Metropolitan University.  Uma abordagem imuno-histoquímica de montagem total, para visualizar a expressão da proteína de neurofilamento no trato biliar extra-hepático em Suncus murinus. É apresentado aqui. Este protocolo pode ser usado para analisar a inervação de todos os órgãos viscerais em S. murinus ou outras espécies. 

Vídeo: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

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 ...

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 ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Oi-C: um método para estudar a arquitetura tridimensional de Genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Usando Monte Whole In situ Hibridação a ligação de Biologia Molecular e Organísmicos

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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 in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

A High Throughput In situ Método de hibridização para caracterizar padrões de expressão de mRNA no Rato Fetal Trato urogenital inferior

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...