Trabalho suportado por concessões de NIH / NIDCD ea Fundação Schmitt para RP.

Este protocolo foi desenvolvido e aperfeiçoado com base no padrão radioativos e não radioativos métodos de hibridização in-situ, anteriormente desenvolvido por nós e outros para detectar uma ou duas espécies transcrição no tecido cerebral 1-7. O protocolo descrito a seguir tem um comprimento total de 2 ou 3 dias, dependendo do número de interrupções processual escolhida pelo usuário final. Todas as etapas detalhadas abaixo devem ser realizados à temperatura ambiente, com exceção da etapa de hibridização riboprobe e pós-hibridação lavagens. As soluções e buffers exigido para todos os passos envolvidos neste método podem ser encontrados no final do presente protocolo. 1. Preparação do tecido e Seccionamento <ol><li> Decapitar e extrair o cérebro do sujeito rapidamente, e colocá-lo em um molde de plástico de tamanho adequado. </li><li> Cobrir o cérebro com Tissue-Tek meio de incorporação e rapidamente coloque o molde de plástico em um banho dry-ice/alcohol para o congelamento rápido. Tecido congelado pode ser armazenado a -80 ° C até o uso. </li><li> Utilizando um criostato, recolher 2 ou 3 seções do cérebro por slide em Superfrost cobrado Além disso slides. Espessura das seções devem ser 10-12 mM. Lâminas podem ser armazenadas a -80 ° C até o uso. </li></ol> 2. Preparação de Sephadex G50 Colunas para Purificação Probe &quot;Colunas Sephadex podem ser adquiridos de fontes comerciais, no entanto, oferecemos a seguir uma alternativa de baixo custo para a geração de colunas, o que será necessário para a purificação da sonda. <ol><li> Quantidade adequada de hidrato de Sephadex G50 em pó com RNase-free, água DEPC-tratados (por exemplo, 2 g de pó em 100 ml de água tratada DEPC-), brevemente solução misturar e armazenar à temperatura ambiente para precipitar o excesso Sephadex G50. </li><li> Remover o sobrenadante (camada superior de água), após precipitação Sephadex G50. </li><li> Repita o processo acima 3-5 vezes. </li><li> Após a lavagem final, re-suspender a Sephadex G50 solução em tampão TE (1:1), e armazenar a 4 ° C até o uso. </li><li> Lugar autoclavado lã de vidro para uma seringa estéril 1 ml e comprimi-lo com o êmbolo para fazer uma camada compacta na parte inferior, e em seguida coloque a seringa em um tubo Falcon 15 ml. </li><li> Misture bem a solução de Sephadex G50 em TE. Encha a seringa / coluna com esta solução. </li><li> Centrifugar a coluna por 30 segundos a 1000 rpm. </li><li> Repita este procedimento até que a coluna é quase completamente preenchida com Sephadex G50 contas. </li><li> Aplicar 200 mL de tampão de lavagem coluna para a coluna e centrifugar por 2 min a 1000 rpm. Descartar por escoamento. </li><li> Aplicar 200 mL de tampão de bloqueio coluna para a coluna e gire-a para 2 min a 1000 rpm. Repita este passo 4-5 vezes para equilibrar coluna. As colunas podem ser armazenados a 4 ° C até o uso se selado com Parafilm. </li></ol> 3. Rotulagem e Purificação de Riboprobes Abaixo detalhamos a geração e purificação de riboprobe único. Para dFISH, a preparação de cada sonda irá envolver a mesma metodologia, exceto que uma das sondas será marcada com digoxigenina (DIG) marcadas UTP enquanto o outro com biotina marcadas UTP. <ol><li> Preparar um concentrado (&gt; 150 ng / mL) e purificado solução linearizada cDNA de interesse para gerar um sentido (controle) ou riboprobes antisense. </li><li> Em um microtubo de 1,5 ml, adicionar 0,5-1 mg de purificada modelo de cDNA, 2 mL de tampão 5X sonda rotulagem, 1 ml de 10X DIG (ou biotina), rotulagem mix, 0,5 mL de RNasin e 1 ml da RNA polimerase adequada , e trazer o volume final da solução para 10 ml com RNAse-free (DEPC-tratados) de água. </li><li> Incubar a solução em um banho de água a 37 ° C por 2 horas. </li><li> Adicionar 1 ml de tRNA (estoque, 20 mcg / mL), e 39 mL de tampão coluna de bloqueio para a solução. </li><li> Prepare o Sephadex G50 coluna para purificação da sonda. Para este fim, adicionar 50 mL tampão de bloqueio para a coluna e gire-a por 10 segundos a 1000 rpm. Repita este passo 2-3 vezes para equilibrar a coluna (ou seja, o total de 50 mL aplicada à coluna são recuperados após o ciclo de centrifugação). </li><li> Aplique a solução sonda para a coluna de Sephadex G50 posição, um tubo de microcentrífuga novo na parte inferior da coluna, e girá-lo por 3 min a 1000 rpm para obter o purificada 50 ml de solução riboprobe. </li><li> Avaliar a qualidade e rendimento de riboprobe rotulados usando um padrão de formaldeído-agarose gel RNA, ou um espectrofotômetro. </li></ol> 4. Pós-fixação Acetilação e Hibridização <ol><li> Remove seções do freezer -80 ° C e permitir uma temperatura ambiente para equilibrar. Posteriormente, incubar seções em um frio, solução de paraformaldeído recém-made de 3% para 5 min. </li><li> Brevemente lavar seções em phosphate solução salina tamponada (PBS) por duas vezes. </li><li> Desidratar seções através de uma série de álcool normal (70, 95 e 100%; 2 min cada), e deixe-os secar ao ar. </li><li> Incubar seções em uma solução de acetilação de 10 min. </li><li> Enxágüe seções 3 vezes em 2X SSPE. </li><li> Desidratar seções, mais uma vez através da série de álcool padrão descrito acima, e permitir-lhes-ar seco. </li><li> Prepare um volume adequado de solução de hibridação e adicionar tanto riboprobes para a solução (concentração da sonda: 1 ng / mL para cada sonda). Volume total da solução de hibridização é determinada com base no número de seções para ser hibridizado (16 ml de solução de hibridização por seção). </li><li> Aplicar volume adequado da solução de hibridização para o tecido e lamínula slides garantir que nenhum ar overlay bolhas no tecido. </li><li> Coloque as lâminas em porta-lâminas de metal. Titular mergulhe em um banho de óleo mineral regulado a 65 ° C durante a noite. Garantir que as lamínulas são virados para cima (ou seja, o aspecto lateral do compartimento de slides deve estar em contato com o fundo do recipiente de óleo mineral). </li></ol> 5. Pós-hibridação Lava <ol><li> No dia seguinte, remova cuidadosamente o suporte de slides do banho de óleo mineral e enxaguá-lo brevemente em clorofórmio para remover o excesso de óleo do slides. </li><li> Coloque as lâminas em uma solução de SSPE 2X de 5-10 min. Lamínulas devem separar de slides enquanto em solução. </li><li> Transferir as lâminas para uma nova solução de SSPE 2x, e mantê-los por 1 hr em temperatura ambiente. </li><li> Transferência de seções em uma solução contendo 2X SSPE além de formamida 50%. A temperatura desta solução deve coincidir com a temperatura utilizada no processo de hibridização durante a noite. Mantenha slides nesta solução por 1,5 hr. </li><li> Transferência de seções para uma solução de SSPE 0,1 x pré-aquecida à mesma temperatura da hibridação. Incubar seções nesta solução por 30 min. Repita este passo para um adicional de 30 min. </li></ol> 6. Detecção e Visualização de Riboprobes <ol><li> Transferência de slides para o buffer TNT em que o peróxido de hidrogênio 0,3% foi adicionado, por 10 min. </li><li> Lave os cortes em TNT buffer, 3 vezes (10 min cada). </li><li> Usando uma caneta DAKO, desenhe um bem ao redor da área que contém as seções do cérebro. Importante, devem ser tomadas precauções para garantir que as seções não seca. </li><li> Aplicar 150 mL de tampão TNB a cada slide, e incubar seções nesta solução por 30 min, em câmara úmida. </li><li> Remoção da solução TNB excesso de slides de inclinação. </li><li> Aplicar 150 mL de solução contendo TNB conjugado com peroxidase de anticorpos anti-DIG e slides armazenar em câmara úmida por 2 horas. Concentração de anticorpos deve ser determinada individualmente para cada transcrição de interesse antes da realização dFISH. </li><li> Lave os cortes em TNT buffer; 3 vezes, 10 min cada. </li><li> Aplicar 150 mL de Alexa 594-conjugados solução tiramida trabalhando para cada slide, e armazená-los em câmara úmida por 1 hora. Esta solução deve ser preparada de acordo com as instruções do fabricante. </li><li> Lave os cortes em TNT buffer; 3 vezes, 10 min cada. </li><li> Seções incubar em tampão TNT para que o peróxido de hidrogênio 0,3% foi adicionado para 10-30 min. </li><li> Lave os cortes em TNT buffer; 3 vezes, 10 min cada. </li><li> Aplicar 150 mL de tampão TNB por lâmina, e mantê-los em câmara úmida por 30 min. </li><li> Remoção da solução TNB excesso de slides de inclinação. </li><li> Adicionar 150 mL de solução contendo TNB conjugado com peroxidase de anticorpos anti-biotina, e slides armazenar em câmara úmida por 2 horas. </li><li> Lave os cortes em TNT buffer; 3 vezes, 10 min cada. </li><li> Aplicar 150 mL de uma Alexa 488-conjugados solução tiramida trabalhando para os slides e seções incubar por 1 hora. Esta solução deve ser preparada de acordo com as recomendações do fabricante. </li><li> Lave os cortes em TNT buffer; 3 vezes, 10 min cada. </li><li> Adicionar 150 mL de solução de Hoechst (1:1000 em TNT buffer) para as seções e mantê-los na câmara úmida por 2 min. </li><li> Lavar as seções em TNT buffer; 3 vezes, 5 min cada. </li><li> Seções lamela com uma média de fluorescência compatível com a montagem (por exemplo, Vectashield ou prolongar Antifade). </li></ol> 7. Resultados representante <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Figura 1" /> Figura 1. Mostramos aqui os resultados obtidos dFISH representante no cérebro de tentilhões-zebra. Mostrados são fotomicrografias obtidas a partir da nidopallium caudomedial (NCM), o análogo songbird do córtex auditivo de mamíferos. Seções do cérebro foram hibridizados com uma riboprobe biotinilado contra parvalbumina (A), um marcador para uma sub-população de neurônios inibitórios, e um DIG marcado ribosonda dirigida contra o dependente de atividade do gene Zenk (B), um marcador confiável para a canção-driven neurônios. C) Sobreposição de (A) e (B) mostra uma população de neurônios inibitórios que são ativadas pela experiência auditiva. Flechas e setas indicam células marcadas exclusivamente com cada um dos dois riboprobes, e asteriscos mostram neurônios representante co-expressar tanto transcrições de interesse. Barra de escala = 25 mm.

<ol>
	<li>Jin, L., Lloyd, 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=In+situ+hybridization:+methods+and+applications.">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li><li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization.">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li><li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+and+signal+amplification:+approaches+to+increase+the+sensitivity+of+in+situ+hybridization.">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li><li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digoxigenin:anti-digoxigenin+(DIG)+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system.">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li><li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+RNA+probes:+facts+and+artifacts.">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li><li>Qian, X., Lloyd, 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=Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization.">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li><li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+song-regulated+genes+in+the+brain+of+songbirds.">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li><li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+participate+in+the+brain's+response+to+birdsong+auditory+stimulation.">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li><li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-induction+of+activity-dependent+genes+in+songbirds.">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li><li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression.">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li><li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol.">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li></ol>

Não há conflitos de interesse declarados.

Nós temos usado esse protocolo para estudar como o cérebro dos vertebrados é neuroquimicamente e funcionalmente organizado, e para determinar como comportamentalmente relevante impacto estímulos sensoriais as máquinas genômica de neurônios no cérebro adulto 80-10. Temos utilizado com sucesso este método em tecido cerebral de camundongos, ratos e pássaros, mas antecipamos que este protocolo será facilmente adaptável às seções do cérebro obtidas a partir de uma variedade de espécies de vertebra...

Soluções <ol><li> Tampão da coluna de lavagem: 10 mM Tris-HCl, 0,15 M NaCl, 0,05 mM EDTA, 50 mcg / mL tRNA, SDS 0,1% em 50 ml de água DEPC-tratada. </li><li> Tampão da coluna de bloqueio: 10 mM Tris-HCl, 50 mM NaCl, 0,1 mM EDTA em 50 ml de água DEPC-tratada. </li><li> TNT buffer: 60 ml de 1 M Tris-HCl, 18 ml de 5 M NaCl e 1,8 ml de Triton X-100 em 600 ml de água tratada DEPC-. </li><li> TNB buffer: 100 mM Tris-HCl, 8,3 mg / mL BSA, 0,15 M NaCl e Triton 3% X-100 em DEPC tratados com água. </li><li> TE buffer: 10 mM Tris-HCl, pH 7,5, acrescido de 1 mM EDTA, pH 7.5. </li><li> Acetilação solução: 2,7 ml de trietanolamina mais 0,5 ml de anidrido acético em 200 ml de água tratada DEPC-. </li><li> Solução de hibridização: formamida 50%, 2X SSPE, 2 mg / mL tRNA, 1 mg / mL BSA e 1 mg / mL poli A em água DEPC-tratada. Ela precisa de 16 l por seção. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

Aqui nós descrevemos uma versão modificada de uma dupla de fluorescência<em&gt; In situ</em&gt; Hibridação método (dFISH) otimizado para a detecção de dois mRNAs de interesse em novas seções cérebro congelado. Nosso grupo tem utilizado com sucesso esta abordagem para o estudo de genes co-regulação. Mais especificamente, temos utilizado este método dFISH para explorar a organização anatômica, propriedades neuroquímicos, eo impacto da experiência sensorial no centro de circuitos sensoriais, em resolução única célula. Este protocolo foi validado no tecido cerebral de camundongos, ratos e pássaros, mas é esperado para ser facilmente adaptável a outras espécies de vertebrados, bem como para uma matriz de não-tecidos neurais. Neste filme, nós fornecemos uma demonstração detalhada dos principais passos deste procedimento.

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Este protocolo envolve um não-radioativo<em&gt; In-situ</em&gt; Processo de hibridação que permite a identificação simultânea de duas espécies de transcrição, com uma resolução única célula, em seções finas do cérebro dos vertebrados.

Fluorescência dupla In situ Hibridação nas Seções cérebro fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K visualizações.  University of Rochester. Este protocolo envolve um não-radioativo In-situ Processo de hibridação que permite a identificação simultânea de duas espécies de transcrição, com uma resolução única célula, em seções finas do cérebro dos vertebrados.

Vídeo: Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Combinando corante lipofílico, In situ Hibridação, imunohistoquímica e histologia

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Neurociência

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Concepção e montagem de um Microdrive Ultra-leve motorizada para doenças crônicas Recordings Neurais em Pequenos Animais

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Spinal Cord Injury completo e Cérebro Dissection Protocolo para Wholemount Subsequente<em&gt; In Situ</em&gt; Hibridização em larval da lampreia de mar

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Detecção de Axonally localizada mRNAs em seções do cérebro Usando de alta resolução<em&gt; In Situ</em&gt; Hibridização

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

Rápido<em&gt; In Situ</em&gt; A hibridação usando sondas Oligonucleotide no cérebro prefixado-paraformaldeído de ratos com síndrome serotoninérgica

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Combinando Duplo Fluorescência<em&gt; In Situ</em&gt; A hibridação com imunomarcação para a detecção da expressão de três genes em secções do cérebro do rato

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localização de Genes de Receptor de Odorantes em Antenas de Locustídeo por RNA<em&gt; In Situ</em&gt; Hibridação

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combinando Hibridização In Situ por Fluorescência Multiplex com Imunohistoquímica Fluorescente em Seções Cerebrais de Camundongos Frescos Congelados ou Fixos

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

Um modelo de rato de infecção cerebral ecohiv

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.