El trabajo apoyado por subvenciones del NIH / NIDCD y la Fundación de Schmitt a la PR.

Este protocolo fue desarrollado y perfeccionado sobre la base de los métodos estándar de hibridación radiactivos y no radiactivos in situ, desarrollado previamente por nosotros y otros para detectar una o dos especies de transcripción en el tejido cerebral 1-7. El protocolo se describe a continuación tiene una longitud total de 2 o 3 días, dependiendo del número de interrupciones del procedimiento elegido por el usuario final. Todos los pasos detallados a continuación se llevarán a cabo a temperatura ambiente, con la excepción de la etapa de hibridación riboprobe y se lava después de la hibridación. Las soluciones y tampones necesarios para todas las etapas de este método se puede encontrar al final de este protocolo. 1. Preparación del tejido y corte <ol><li> Decapitar y extracto de cerebro de sujetos en rápido, y colocarlo en un molde de plástico de tamaño adecuado. </li><li> Cubren el cerebro con Tissue-Tek medio de inclusión y rápidamente colocar el molde de plástico en un baño de dry-ice/alcohol de congelación rápida. El tejido congelado se puede almacenar a -80 ° C hasta su uso. </li><li> El uso de un criostato, recoger dos o tres secciones del cerebro por diapositiva en Superfrost Plus diapositivas cargado. Espesor de las secciones debe ser 10-12 micras. Las diapositivas se pueden almacenar a -80 ° C hasta su uso. </li></ol> 2. Preparación de Sephadex G50 columnas para la purificación de la sonda &quot;Columnas de Sephadex se pueden adquirir de fuentes comerciales, sin embargo, a continuación una alternativa de bajo costo para la generación de columnas, que se requiere para la purificación de la sonda. <ol><li> Cantidad adecuada de hidratos de Sephadex G50 en polvo con RNasa libre, agua tratada con DEPC (por ejemplo, 2 g de polvo en 100 ml de agua tratada con DEPC), una breve solución de mezclar y almacenar a temperatura ambiente para precipitar el exceso de Sephadex G50. </li><li> Aspirar el sobrenadante (la capa superior de agua) después de Sephadex G50 precipitación. </li><li> Repita el proceso por encima del 3 - 5 veces. </li><li> Después del lavado final, volver a suspender las Sephadex G50 solución en buffer TE (1:1), y se almacenan a 4 ° C hasta su uso. </li><li> Coloque el autoclave lana de vidrio en una jeringa estéril de 1 y comprimir con el émbolo para hacer una capa compacta en la parte inferior, y luego colocar la jeringa en un tubo Falcon de 15 ml. </li><li> Mezcle bien la solución de Sephadex G50 en TE. Llene la jeringa / columna con esta solución. </li><li> Centrifugar la columna durante 30 segundos a 1000 rpm. </li><li> Repita este procedimiento hasta que la columna está casi completamente lleno de Sephadex G50 cuentas. </li><li> Aplicar 200 l de tampón de columna de lavado a la columna y centrifugar durante 2 min a 1000 rpm. Desechar el filtrado. </li><li> Aplicar 200 l de tampón de columna de bloqueo a la columna y girar durante 2 min a 1000 rpm. Repita este paso 4-5 veces para equilibrar la columna. Las columnas pueden ser almacenadas a 4 ° C hasta su uso si sellado con Parafilm. </li></ol> 3. Etiquetado y purificación de ribosondas A continuación se detallan la generación y purificación de un riboprobe sola. Para dFISH, la preparación de cada sonda implicará la misma metodología, excepto que una de las sondas serán marcadas con digoxigenina (DIG)-etiquetados UTP mientras que el otro con biotina-etiquetados UTP. <ol><li> Prepare una solución concentrada (&gt; 150 ng / l) y se purifica solución linealizada cDNA de interés para generar uno u otro sentido (control) o riboprobes antisentido. </li><li> En un tubo de microcentrífuga de 1,5 ml, agregar 0,5-1 g de agua purificada plantilla de cDNA, 2 l de tampón 5X etiquetado sonda, 1 l de DIG 10 veces (o biotina)-etiquetado mezcla, 0,5 l de RNasin y 1 l de la correspondiente ARN polimerasa , y llevar el volumen final de la solución a 10 ml con RNasa libre (tratada con DEPC) de agua. </li><li> Incube la solución en un baño de agua a 37 ° C durante 2 horas. </li><li> Añadir 1 l de tRNA (stock; 20 mg / l), y 39 l de tampón de columna de bloqueo a la solución. </li><li> Prepare la columna de Sephadex G50 para la purificación de la sonda. Con este fin, se añaden 50 l buffer de bloqueo a la columna y girar durante 10 segundos a 1000 rpm. Repita este paso 2-3 veces para equilibrar la columna (es decir, la totalidad de 50 l aplicada a la columna se recuperan después de un ciclo de centrifugado). </li><li> Aplique la solución de prueba a la columna de Sephadex G50, la posición de un tubo de microcentrífuga nuevo en la parte inferior de la columna, y girar durante 3 minutos a 1000 rpm para obtener el purificada 50 l de solución riboprobe. </li><li> Evaluar la calidad y el rendimiento de riboprobe etiquetados utilizando una norma de formaldehído-agarosa ARN gel, o un espectrofotómetro. </li></ol> 4. Posterior a la fijación, acetilación y de hibridación <ol><li> Eliminar secciones de la 80 ° C congelador y permitir una temperatura ambiente que se equilibre. Posteriormente, se incuban las secciones de un frío, recién hechas solución de paraformaldehído al 3% durante 5 min. </li><li> Brevemente enjuague secciones en phosphate solución salina (PBS) dos veces. </li><li> Deshidratar secciones a través de una serie estándar de alcohol (70, 95 y 100%, 2 minutos cada uno), y dejar que se seque al aire. </li><li> Incubar las secciones en una solución de acetilación de 10 min. </li><li> Enjuague las secciones 3 veces en 2 veces la SSPE. </li><li> Deshidratar secciones, una vez más a través de la serie estándar de alcohol se ha descrito anteriormente, y permitir que se seque al aire. </li><li> Preparar un volumen adecuado de solución de hibridación y añadir tanto riboprobes a la solución (concentración de la sonda: 1 ng / l para cada sonda). Volumen total de la solución de hibridación se determina en función del número de secciones que se han hibridado (16 l de solución de hibridación por sección). </li><li> Aplicar volumen adecuado de la solución de hibridación en las diapositivas de tejidos y cubreobjetos asegurar que el aire no superposición de las burbujas de los tejidos. </li><li> Coloque los portaobjetos en un soporte de diapositivas de metal. Titular de sumergirse en un baño de aceite mineral a los 65 ° C durante la noche. Asegúrese de que los cubreobjetos son hacia arriba (es decir, la parte lateral del soporte de diapositivas debe estar en contacto con el fondo del recipiente de aceite mineral). </li></ol> 5. La hibridación después de Lava <ol><li> Al día siguiente, retirar con cuidado el soporte de diapositivas del baño de aceite mineral y brevemente se aclare en cloroformo para eliminar el exceso de grasa de las diapositivas. </li><li> Coloque los portaobjetos en una solución de 2X PES durante 5-10 minutos. Cubreobjetos debe desprenderse de diapositivas, mientras que en la solución. </li><li> Transferencia de las diapositivas de una nueva solución de 2X SSPE, y mantenerlos durante 1 hora a temperatura ambiente. </li><li> Transferencia de secciones en una solución que contiene la SSPE 2X más el 50% de formamida. La temperatura de esta solución debe coincidir con la temperatura utilizada en el procedimiento de hibridación noche a la mañana. Mantenga las diapositivas en esta solución durante 1,5 horas. </li><li> Transferencia de las secciones a una solución 0.1X PEES pre-calentado a la misma temperatura de la etapa de hibridación. Incubar las secciones en esta solución durante 30 minutos. Repita este paso para un período adicional de 30 min. </li></ol> 6. Detección y visualización de ribosondas <ol><li> Transferencia de diapositivas para amortiguar TNT a las que 0,3% de peróxido de hidrógeno se ha añadido, durante 10 minutos. </li><li> Lávese las secciones de TNT buffer, 3 veces (10 minutos cada uno). </li><li> El uso del lápiz Dako, dibujar un pozo en la zona que contiene las secciones del cerebro. Es importante destacar que se debe tener precaución para que los artículos no se sequen. </li><li> Aplicar 150 l de tampón TNB a cada diapositiva, e incubar las secciones en esta solución durante 30 minutos, en una cámara húmeda. </li><li> Retire el exceso de solución TNB diapositivas inclinación. </li><li> Aplicar 150 ml de solución contiene TNB conjugado con peroxidasa anti-DIG anticuerpos, y se desliza almacenar en una cámara húmeda durante 2 horas. Concentración de anticuerpos debe determinarse individualmente para cada una transcripción de interés antes de realizar dFISH. </li><li> Lávese las secciones de TNT buffer, 3 veces, 10 minutos cada uno. </li><li> Aplicar 150 ml de Alexa 594-conjugado solución tyramide de trabajo para cada diapositiva, y almacenarlos en una cámara húmeda durante 1 hora. Esta solución debe prepararse de acuerdo con las instrucciones del fabricante. </li><li> Lávese las secciones de TNT buffer, 3 veces, 10 minutos cada uno. </li><li> Secciones se incuban en un tampón de TNT a las que 0,3% de peróxido de hidrógeno se ha añadido durante 10 - 30 min. </li><li> Lávese las secciones de TNT buffer, 3 veces, 10 minutos cada uno. </li><li> Aplicar 150 l de tampón TNB por diapositiva, y mantenerlas en una cámara húmeda durante 30 minutos. </li><li> Retire el exceso de solución TNB diapositivas inclinación. </li><li> Añadir 150 ml de solución de TNB que contienen conjugado de peroxidasa anti-biotina de anticuerpos, y se desliza almacenar en una cámara húmeda durante 2 horas. </li><li> Lávese las secciones de TNT buffer, 3 veces, 10 minutos cada uno. </li><li> Aplicar 150 ml de un Alexa 488-conjugado solución tyramide de trabajo a las diapositivas y las secciones se incuban durante 1 hora. Esta solución debe prepararse de acuerdo con las recomendaciones del fabricante. </li><li> Lávese las secciones de TNT buffer, 3 veces, 10 minutos cada uno. </li><li> Añadir 150 ml de solución de Hoechst (1:1000 en buffer de TNT) a las secciones y los mantienen en la cámara húmeda durante 2 min. </li><li> Lave las secciones de TNT buffer; 3 veces, 5 minutos cada uno. </li><li> Cubreobjetos secciones con un medio de fluorescencia compatible con el montaje (por ejemplo, Vectashield o prolongar Antifade). </li></ol> 7. Resultados representante <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Figura 1" /> Figura 1. Mostramos aquí los resultados obtenidos dFISH representante en el cerebro pinzón cebra. Se muestran microfotografías obtenidas de la caudomedial nidopallium (NCM), el análogo de pájaro cantor de la corteza auditiva de mamíferos. Secciones del cerebro se hibridó con una riboprobe biotinilado dirigido contra parvalbúmina (A), un marcador de una subpoblación de neuronas inhibitorias, y una marcada con DIG riboinvestigación dirigida contra el Zenk dependiente de la actividad del gen (B), un marcador fiable para impulsada canción neuronas. C) Superposición de (A) y (B) muestra una población de neuronas inhibitorias que se activan por la experiencia auditiva. Flechas y puntas de flecha indican las células marcadas exclusivamente con cada uno de los dos riboprobes y asteriscos muestran las neuronas representante co-expresión de ambas transcripciones de interés. Barra de escala = 25 micras.

<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>

Hemos utilizado este protocolo para estudiar cómo el cerebro de los vertebrados es neuroquímico y funcionalmente organizada, y para determinar la conducta relevante impacto estímulos sensoriales de la maquinaria del genoma de las neuronas en el cerebro adulto 80-10. Hemos utilizado con éxito este método en el tejido cerebral de ratones, ratas y pájaros, pero anticipamos que este protocolo se adapta fácilmente a las secciones del cerebro obtenidas de una gran variedad de especies de vertebrados y, quizás, los tejidos no neuronales. Controles clave necesarios para la obtención de resultados fiables y óptima de la señal se han detallado extensamente por nuestro grupo en otros lugares 11, 12.

Soluciones <ol><li> Columna de tampón de lavado: 10 mM Tris-HCl, NaCl 0,15 M, 0,05 mM EDTA, 50 mg / l tRNA, SDS 0,1% en 50 ml de agua tratada con DEPC. </li><li> Columna de tampón de bloqueo: 10 mM Tris-HCl, 50 mM NaCl, 0,1 mM EDTA en 50 ml de agua tratada con DEPC. </li><li> TNT búfer: 60 ml de 1 M Tris-HCl, 18 ml de 5 M NaCl y 1,8 ml de Triton X-100 en 600 ml de agua tratada con DEPC. </li><li> TNB búfer: 100 mM Tris-HCl, 8,3 mg / l de BSA, NaCl 0,15 M y el 3% Triton X-100 en agua tratada con DEPC. </li><li> Tampón TE: 10 mM Tris-HCl, pH 7,5 y 1 mM EDTA, pH 7,5. </li><li> Solución de la acetilación: 2.7 ml de trietanolamina más 0,5 ml de anhídrido acético en 200 ml de agua tratada con DEPC. </li><li> Solución de hibridación: formamida al 50%, 2x SSPE, 2 mg / l tRNA, 1 mg / l de BSA y 1 mg / l poli A en el agua tratada con DEPC. Se necesita 16 litros por sección. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

Aquí se describe una versión modificada de un fluorescente doble<em&gt; In situ</emHibridación&gt; (dFISH) método optimizado para la detección de dos mRNAs de interés en nuevas secciones del cerebro congelado. Nuestro grupo ha utilizado con éxito este enfoque para el estudio de genes co-regulación. Más específicamente, hemos utilizado este método para explorar dFISH la organización anatómica, propiedades neuroquímicas, y el impacto de la experiencia sensorial en el centro de los circuitos sensoriales, con una resolución única célula. Este protocolo ha sido validado en el tejido cerebral de ratones, ratas y pájaros, pero se espera que sea fácilmente adaptable a otras especies de vertebrados, así como a una amplia gama de tejidos no neuronales. En esta película una demostración detallada de las principales etapas de este procedimiento.

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 consiste en un no-radiactivos<em&gt; In-situ</emProcedimiento&gt; hibridación que permite la identificación simultánea de dos especies de transcripción, en una resolución única célula, en secciones finas de cerebro de los vertebrados.

Doble fluorescencia In situ La hibridación en las secciones del cerebro 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 ...

double-fluorescence-in-situ-hybridization-in-fresh-brain-sections

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K vistas.  University of Rochester. Este protocolo consiste en un no-radiactivos In-situ hibridación que permite la identificación simultánea de dos especies de transcripción, en una resolución única célula, en secciones finas de cerebro de los vertebrados.

Video: 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.

La combinación de colorantes lipofílicos, In situ</emHibridación>, inmunohistoquímica y la histología

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

Investigación

Neurociencias

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.

Diseño y montaje de un Microdrive ultraligero motorizado para crónicos Grabaciones neuronales en Pequeños Animales

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.

Lesiones de la Médula Espinal completo y Protocolo Disección del cerebro por Wholemount posterior<em&gt; In Situ</em&gt; La hibridación en larvas lamprea 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.

La detección de ARNm axón localizado en las secciones cerebrales Uso de alta resolución<em&gt; In Situ</em&gt; La hibridación

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; La hibridación usando sondas de oligonucleótidos en Cerebro paraformaldehído-prefijo de ratas con síndrome de serotonina

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.

La combinación de doble fluorescencia<em&gt; In Situ</em&gt; La hibridación con Immunolabelling para la detección de la expresión de tres genes en las secciones de cerebro de ratón

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.

Localización de Genes Receptor Odorante en Antenas Locustinas por RNA<em&gt; In Situ</em&gt; Hibridación

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

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Aislamiento de los capilares cerebrales del cerebro humano fresco tejido

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Doble inyección directa de sangre en la Cisterna Magna como modelo de hemorragia subaracnoidea

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

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.

Combinación de hibridación in situ de fluorescencia múltiple con inmunohistoquímica fluorescente en secciones de cerebro de ratón frescas, congeladas o fijas

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