Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
Uploaded: 2017-06-15T12:30:00.000+00:00
Duration: 7 min 23 s
Description: 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

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>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+study+of+the+pancreas+in+the+house+musk+shrew,+Suncus+murinus,+with+special+reference+to+the+blood+supply+and+innervation.">Anatomical study of the pancreas in the house musk shrew, Suncus murinus, with special reference to the blood supply and innervation.</a> Anat. Rec. A Discov. Mol. Cell Evol. Biol. 273 (1), 630-635 (2003).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+the+innervation+of+the+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. 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. Embryol. 45 (3), 184-188 (2016).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofilament+proteins+in+axonal+regeneration+and+neurodegenerative+diseases.">Neurofilament proteins in axonal regeneration and neurodegenerative diseases.</a> Neural Regen. Res. 7 (8), 620-626 (2012).</li><li>Wyatt, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+the+sphincter+of+Oddi+to+the+stomach,+duodenum+and+gall-bladder.">The relationship of the sphincter of Oddi to the stomach, duodenum and gall-bladder.</a> J. Physiol. 193 (2), 225-243 (1967).</li><li>Mawe, G. M., Kennedy, A. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+nerve+cells+from+the+duodenum+to+the+sphincter+of+Oddi+and+gallbladder+of+the+Australian+possum.">Projections of nerve cells from the duodenum to the sphincter of Oddi and gallbladder of the Australian possum.</a> Gastroenterology. 104 (1), 130-136 (1993).</li><li>White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+immunohistochemistry+for+revealing+complex+brain+topography.">Wholemount immunohistochemistry for revealing complex brain topography.</a> J. Vis. Exp. (62), e4042 (2012).</li></ol>

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 ...

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><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>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 ( <strong cl...

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 ...

using-whole-mount-immunohistochemical-method-to-study-innervation

Universidad de Cartagena UDEC

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

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining para Visualize Murino desenvolvimento do sistema nervoso entérico

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

Biologia do Desenvolvimento

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

In vivo localização de imunofluorescência para avaliação de anticorpo terapêutico e diagnóstico biodistribuição em pesquisa de câncer

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Pesquisa do câncer

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Determinando a densidade do ducto biliar no fígado do rato

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Neurociência

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualização das células intersticiais de Cajal Network (ICC) em ratos

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biologia

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Imunocoramento Secções biocitina-preenchidos e processados ​​para neuroquímicos Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Imunologia e Infecção

Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

Source: Michael S. Lee1 and Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, Maryland 21201
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques used to visualize the expression and localization of specific antigens using antibodies. The first published use of IHC was in 1941 when Albert Coons used the technique to visualize the presence of pneumococcal antigen in tissue sections from mice infected with Pneumococcus (1). The name, immunohistochemistry, is derived from the roots &#34;immuno-,&#34; in reference to antibodies, and &#34;histo-,&#34; in reference to the tissue sections used in IHC. The root &#34;cyto-&#34; in immunocytochemistry highlights the key difference between ICC and IHC. Whereas IHC uses sections of whole tissue, ICC uses cells that have been isolated from tissue or grown in culture. The difference in samples used means sample preparation technically differs between IHC and ICC, but otherwise the protocols for ICC and IHC are identical and one will find the terms are frequently used interchangeably.
In both IHC and ICC, antibodies with either chemical or fluorescent tags, such as peroxidase or rhodamine, respectively, are used to visualize the distribution of any antigen of interest through specific binding of the tagged antibody to the antigen. In the case of IHC, thin slices of tissue are immobilized on a slide to maintain the structure of the tissue before being stained, allowing the visualization of antigens in the context of whole tissues (Figure 1). In the case of ICC, cells are distributed evenly on a slide before being stained, allowing the visualization of antigen distribution within individual cells but not within the structure of any specific tissue. Due to the similarities between the two protocols, this protocol will focus on IHC to address the additional complexities of sample preparation involved in IHC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10498/10498fig1.jpg" /> 
Figure 1: Outline of IHC Protocol. Visual outline of an IHC protocol for paraffin-embedded tissue dissected from a mouse. This protocol uses a biotinylated secondary antibody and strepavidin-HRP to visualize the location of antibody binding. Other options, such as fluorescently tagged antibodies, are also possible. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/10498/10498fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The first major decision when performing IHC is how to prepare the tissue sections in order to maintain the structure of the tissue throughout the staining process. The two main choices are formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. There is no simple answer as to which method to use as it depends on what downstream analysis will be conducted. Formalin-fixation of paraffin embedded tissues is generally thought to better preserve tissue morphology for optimal imaging while freezing fresh tissue can preserve protein function for subsequent assays outside of IHC. In addition, fresh frozen tissue sections have been shown to be more suitable for gene expression analysis (2). A third consideration is whether or not the antibodies for your antigen of interest are suitable for either fixed or frozen tissue sections, as some antibodies have only been optimized for a specific type of section and may not work for others. Finally, one also needs to determine how long they need to store the tissue sections, as fresh frozen samples must be kept at -80&#176;C and may not last beyond one year while fixed sections can be stored for much longer at room temperature. These are a few of the major considerations for determining whether to use formalin-fixed sections of paraffin-embedded tissue or fresh sections of frozen tissue. Ultimately, if one has enough tissue, it may be best just to have some of both.
In this experiment, we set out to determine if cyclin D1 expression was increased in enlarged spleens from a spontaneous mouse model of lymphoma development. Splenic tissue samples were first isolated from either wild-type mice, transgenic mice that do not have lymphoma, or transgenic mice that have spontaneously developed lymphoma. The spleen tissue samples were fixed in paraformaldehyde, embedded in paraffin, sectioned, stained using a mouse anti-cyclin D1 primary antibody followed by a horse anti-mouse secondary antibody, and developed using 3,3-diaminobenzidine (DAB). The sections were then counterstained in Harris Hematoxylin Solution and then the sections were imaged at 20X magnification.
Reagents
Paraffin-Embedded Sections
<ol type="a">
	<li>4% Paraformaldehyde (PFA)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100%, 95%, 80%, 75%, and 50%). Can be diluted from 100% stock using double distilled water (ddH2O)</li>
	<li>Xylene</li>
	<li>IHC compatible glass slide to ensure tissue section remains attached throughout the entire procedure. IHC compatible glass slides have a specialized coating and are readily available from multiple retailers. If performing ICC, use a chambered slide. Chambered slides allow for cells to be seeded in the chambers and placed in the incubator until cells attach to the slide and reach proper confluency, at which point the chambers can be removed and staining can proceed in the same manner as IHC.</li>
	<li>Paraffin</li>
	<li>0.3% Hydrogen peroxide (H2O2)/methanol: To prepare, add 1 mL 30% H2O2 to 99 mL methanol. Store at -20&#176;C</li>
	<li>Antigen retrieval buffer: IHC citrate buffer pH 6.0</li>
</ol>
Fresh Frozen Sections
<ol type="a">
	<li>Optimal cutting temperature (OCT) embedding compound</li>
	<li>Optimal fixative: 4% PFA or acetone that has been cooled to -20&#176;C</li>
</ol>
Staining
<ol type="a">
	<li>Blocking buffer: Should be determined by user. One example is horse serum diluted in 1X PBS</li>
	<li>Diluted primary antibody: see manufacturer specifications</li>
	<li>Diluted biotinylated secondary antibody: see manufacturer specifications</li>
	<li>Diluted avidin-Horseradish Peroxidase (HRP): Only for peroxidase visualization. See manufacturer specifications.</li>
	<li>DAB or another compatible substrate</li>
	<li>Counterstain (optional)</li>
	<li>Ethanol (anhydrous denatured, histological grade 100% and 95%)</li>
	<li>Xylene</li>
	<li>Organo/Limonene Mount</li>
</ol>

Immunohistochemistry &amp; Immunocytochemistry: Tissue Imaging

Imunohistoquímica e imunocitoquímica: imageamento de tecidos via microscopia de luz

Source: Michael S. Lee1 and Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and ...

Education

JoVE Science Education

Biologia Avançada

Imunologia

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Following anesthetization as described in the text protocol, prepare for the tail vein inoculation of the antibody solutions. Disinfect the tail of the animal by wiping three times with an alcohol wipe. Dilate the tail veins by warming with a heat pad for approximately 30 seconds; avoid heating the entire animal. Using a 27-gauge tail vane catheter insert the butterfly needle into one of the two lateral tail veins.
Visualize blood backflow into the catheter to ensure that the needle is properly placed so that the solutions will fully enter the animal versus being captured in the tail.
Use a piece of surgical tape to carefully fix the tail with the inserted needle to the stage. Flush the catheter with 25 microliters of sterile phosphate-buffered saline. Then, inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 microliters of sterile PBS. Remove the needle from the tail, and apply pressure to stop any bleeding.
Perform humane euthanasia of the mouse, as described in the text protocol, and lay the mouse in the supine position. Use surgical scissors and forceps to excise tumor tissues. Use forceps to grasp only the outer layer of skin between the set of mammary glands closest to the tail, and to make a small incision with a pair of surgical scissors. Introduce the closed scissors into the cut, and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane, keeping it intact.
Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the third and fourth mammary glands, make a horizontal cut across the abdomen, to allow retraction of the skin and visualization of the mammary glands. Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors.
Place excised tissues into tissue disposable base molds that are pre-labeled and filled with optimal cutting temperature embedding medium. Quickly freeze the molds by placement on dry ice. In order to study off-target delivery, excise other tissues or organs of interest. Using a cryostat, section frozen tissue blocks at 10-micron thickness, and place adjacent sections onto pre-labeled adhesion glass slides.
Rinse frozen tissue slides with room temperature PBS for 5 minutes to remove the embedding medium. Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. Fix the tissue sections with 4% paraformaldehyde solution for 5 minutes. After rinsing the slides in PBS for 5 minutes, permeabilize tissue sections with 0.5% Triton X-100 in PBS for 15 minutes. Rinse the slides again in PBS for 5 minutes.
Block the tissues with PBS containing 3% weight per volume bovine serum albumin and 5% volume per volume goat serum for 1 hour at room temperature. After rinsing the slides in PBS for 5 minutes, incubate the sections with record-keeping primary antibodies, such as a common nuclear, vascular, or cytoplasmic marker. Here, rat anti-mouse CD31 is used at a 1 to 100 dilution in the blocking solution. The slides with primary antibody are left overnight at 4 degrees Celsius, protected from dehydration on a slide tray.
Following incubation, rinse the slides in PBS for 5 minutes three times. Change the PBS each time. Incubate the slides with secondary antibodies to label the primary antibodies. For this application, visualize anti-B7-H3 antibody using AlexaFluor-546 conjugated goat anti-rabbit antibody, and visualize CD31 with AlexaFluor-488 goat anti-rat secondary antibody in blocking solution. Protect the slides from light and dehydration on a slide tray for 1 hour at room temperature.
After rinsing the slides in PBS, as before, apply one drop of the mounting medium into the center of the tissue slice. Carefully place a coverslip avoiding entrapment of air bubbles. Seal the edges of the coverslip with clear nail polish and allow to dry. Proceed with confocal microscopy imaging and quantitative image analysis, as described in the text protocol.

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em

Resumo

Explore mais vídeos

Capítulos neste vídeo

Immunostaining para Visualize Murino desenvolvimento do sistema nervoso entérico

In vivo localização de imunofluorescência para avaliação de anticorpo terapêutico e diagnóstico biodistribuição em pesquisa de câncer

Determinando a densidade do ducto biliar no fígado do rato

In Situ Ca ²⁺ Imagem do Sistema Nervoso Entérico

Visualização das células intersticiais de Cajal Network (ICC) em ratos

Imunocoramento Secções biocitina-preenchidos e processados para neuroquímicos Markers

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

Imunohistoquímica e imunocitoquímica: imageamento de tecidos via microscopia de luz

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Um método de localização de imunofluorescência in vivo para estudar a biodistribuição de anticorpos

Usando um método imunohistoquímico de montagem total para estudar a preservação do trato biliar em

Resumo

Explore mais vídeos

Capítulos neste vídeo

Immunostaining para Visualize Murino desenvolvimento do sistema nervoso entérico

In vivo localização de imunofluorescência para avaliação de anticorpo terapêutico e diagnóstico biodistribuição em pesquisa de câncer

Determinando a densidade do ducto biliar no fígado do rato

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Visualização das células intersticiais de Cajal Network (ICC) em ratos

Imunocoramento Secções biocitina-preenchidos e processados ​​para neuroquímicos Markers

Imagem latente intravital de linfócitos intraepithelial no intestino pequeno murine

Imunohistoquímica e imunocitoquímica: imageamento de tecidos via microscopia de luz

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Um método de localização de imunofluorescência in vivo para estudar a biodistribuição de anticorpos

In Situ Ca ²⁺ Imagem do Sistema Nervoso Entérico

Imunocoramento Secções biocitina-preenchidos e processados para neuroquímicos Markers