Este trabajo fue apoyado por el Instituto Nacional de Salud Subvenciones R01HL089462 a SP.

1. Los liposomas catiónicos Preparación <ol><li> Autoclave un matraz limpio para ser utilizado para la preparación lipososmes. </li><li> Preparar soluciones madre: <ol class="lalpha"><li> - Disolver 200 mg de bromuro de dimetil dioctadecil amonio (Doab) en 10 ml de cloroformo usando una botella de vidrio pequeño. </li><li> - Disolver 200 mg de colesterol en 10 ml de cloroformo usando una botella de vidrio pequeño. </li><li> - Preparar y esterilizar en autoclave para esterilizar el 5% de glucosa en 100 ml de ARN / ADN-asa agua destilada libre. </li></ol></li><li> Enjuagar el matraz con cloroformo. Añadir 5-10 ml al matraz, remolino, y dejar que repose durante unos minutos. Mantener el matraz cubierto con papel de aluminio en todo momento. Vacíe el cloroformo del matraz. </li><li> Preparación de liposomas: <ol class="lalpha"><li> - Añadir 315 l de Doab solución madre en el matraz de fondo redondo. </li><li> - Añadir 200 l de solución madre de colesterol al matraz. </li><li> - Añadir 9,5 ml de cloroformo al matraz unand mezcla por turbulencia. </li></ol></li><li> Ajuste el rotavapor a 37 ° C, 100 revoluciones por minuto (rpm) o más (100-120 rpm). El rotavapor se debe conectar a un control de vacío de la bomba de agua con un tubo de succión conectado correctamente. </li><li> Conecte el tanque de gas argón y el rotavapor. </li><li> Coloque el matraz al rotavapor y ajustar la configuración óptima antes de bajar en el baño de agua. Abra la primera válvula para el argón completamente en sentido antihorario. Siempre abrir y cerrar la válvula de primera. La segunda válvula controla la presión de argón que sopla en al matraz. La presión debe ser siempre suave. </li><li> Baje el rotavapor en el baño de agua, suficiente para el frasco de tocar el agua sin estar completamente sumergida y encienda la velocidad. </li><li> Espere hasta que todas las cloroformo se evapora, a unos 10 min. A continuación, detenga la máquina. </li><li> Apague la velocidad rotavapor y las válvulas del tanque de argón. Ahora el matraz debe ser eliminado. </li><li> Añadir 2 ml de glucosa al 5% a the matraz para disolver los lípidos. </li><li> Raspe el fondo del frasco a fondo con 1 ml punta de la pipeta para disolver los lípidos. Después de rascarse, eliminar cualquier resto de grasa de la punta de 1 ml en el frasco, si es necesario, con un segundo punta. </li><li> Incubar la solución obtenida en el baño de agua 42 ° C, mientras que lentamente agitación (15 rpm) al matraz. </li><li> Llene el tanque de ultrasonidos con agua fría. Someter a ultrasonidos la solución durante al menos 20-30 minutos con el matraz mantenido y la parte inferior apenas tocando el agua. </li><li> Calentar el matraz en el baño de agua del evaporador rotatorio durante 20 min a 42 ° C, a velocidad cero, con el fondo redondo se sumerge en agua, esta vez. </li><li> Filtrar los liposomas a través de un 0,47 micras y, posteriormente, filtros de jeringa de 0,22 micrones. </li><li> El uso de un pequeño extrusor, filtrar los liposomas a través de una membrana de 50 nm para generar una población homogénea de vesículas de liposomas unilamelares. </li><li> La transferencia de los liposomas a un tubo Eppendorf y colocarlo en hielo. </li></ol><html> 2. Preparar los liposomas: siRNA ITSN-1 Complejos <ol><li> Resuspender el ratón en-blanco ITSN ARNsi en tampón de ARNsi que contiene 20 mM de HEPES y cloruro de sodio 150 mM, pH 7,4, a una concentración final de 2 mg / l. Guárdelo en hielo antes de la mezcla. </li><li> Preparación de los liposomas: ITSN complejos de siRNA en una proporción de 8 nmol de liposomas: 2 mu g de ARN (o 400 nmol de liposomas: 100 g ARNsi ITSN). En el presente estudio, se añadieron 50 l de ITSN siRNA de la solución madre (50 mM) a 100 l de liposomas recién preparadas usando un ARN / ADN-asa libre de Eppendorf tubo. Tenga en cuenta que la concentración de ITSN ARNsi es muy importante, ya que un máximo de 150 l de mezcla puede ser inyectada bilateralmente y retro-orbital en el ratón a la vez. </li><li> Mantenga la mezcla en hielo mientras se espera para inyectar a los ratones. </li></ol> 3. Ratón inyección en la vena retro-orbital (ángulo interno de la órbita del ojo) jove_content &quot;&gt; Todos los experimentos con ratones se aprobaron y se realizan de acuerdo con las directrices de la Universidad Rush Institucional Cuidado de Animales y el empleo. <ol><li> Se anestesia el ratón por inyección intra-peritoneal de un mg / kg de ketamina peso 50 corporal y 5 mg / kg de xilazina peso corporal (mezcla disponible comercialmente). Dependiendo del peso de los animales y la edad, ajuste el volumen y la concentración de los anestésicos. Los ratones utilizados en el estudio son los ratones CD1 ratones machos, de 6-8 semanas de edad y pesan alrededor de 25 gramos. Las jeringas son de tuberculina de 1 ml tipo con la aguja puesta. </li><li> Mantenga orejas de ratón con una mano y gire el ratón sobre un lado para exponer el ángulo interno del ojo. Apunte medial de la plica semilunar, evite tocar el globo ocular. </li><li> Acérquese a la carúncula lagrimal del ojo que sostiene la jeringa que contiene la mezcla en un ángulo de 45 grados en los tres ejes. </li><li> Inyecte lentamente la mezcla y dejar que el ratón para recuperarse con el lado inyectado arriba. Si un líquido grandeformas de gotas en el lugar de inserción de la aguja, retraer la aguja, la succión de nuevo la gota en la jeringa y vuelva a intentarlo. Las inyecciones repetidas se hacen mejor por la alternancia de los ojos, para permitir la recuperación perfecta. Si no está seguro, esta técnica se puede comprobar mediante la inyección de colorante azul (50 l, 0,5% de cristal violeta en metanol) y la exposición de los pulmones de los ratones para observar la vasculatura azul. </li><li> Los ratones en este estudio se inyectaron cada día 3 ª durante 3 semanas sin víctimas mortales o efectos adversos. </li></ol> 4. Ratón de perfusión pulmonar y recogida de tejidos para estudios bioquímicos <ol start="0"><li> Establecer la bomba peristáltica para funcionar a 1,5 ml / min. </li><li> Preparar el entorno: ventilador, mesa de operaciones, instrumentos, hisopos de algodón estériles, q-tips, cuerdas, vasos, agua destilada, salina equilibrada de Hank, trazador. </li><li> Se anestesia el ratón mediante inyección intraperitoneal de la mezcla anestésica (100 mg / kg de ketamina peso corporal y 10 mg / kg de xilazina peso corporal). </li&gt; <li> A nivel del cuello, eliminar las glándulas salivales y exponer la tráquea. </li><li> Comience con la laparotomía, cortar el diafragma, y ​​seguir con la toracotomía, la exposición de los pulmones y el corazón. </li><li> Retire el timo. </li><li> Con el microscopio óptico para una mayor precisión, cateterización de la arteria pulmonar. </li><li> Haga traqueotomía y entubar el ratón usando el traqueostoma. Ajuste el ventilador a una velocidad de 150 golpes / min y el volumen de carrera de 150 l. </li><li> Como salida, abra la aurícula izquierda. </li><li> Perfundir la vasculatura pulmonar del ratón libre de sangre durante 5 min, usando la bomba peristáltica y solución de Hank calentado a 37 ° C. </li><li> Perfundir el trazador (8 nm de oro-albúmina 16) para 10 min adicionales a la misma velocidad de flujo. </li><li> Enjuague el trazador no unido por 5 min de perfusión pulmonar con solución de Hank. </li><li> Siga con la fijación in situ de los pulmones por 10 min de perfusión de 4% (peso / vol) de formaldehído, 2,5% de glutaraldehído y ácido tánico al 1% en 0,1 TUBOStampón, pH 7,2. </li><li> Recoge los pulmones, retire el exceso de tejido, cortar pequeños bloques (3/3 mm), y colocarlos en un vial de centelleo de marcado que contiene 2 ml de la mezcla de fijador. </li></ol> Los ratones caducarán completamente anestesiada durante el procedimiento. 5. Ratón pulmón Procesamiento de tejido de EM <ol><li> Preparar soluciones madre: 1% Palade OsO4, acetato de veronal y tampón Kellenberger. <ol class="lalpha"><li> - 1% Palade-OSO tampón 4 contiene: 1 ml de caldo de veronal de etilo, 1,25 ml de 4% OSO 4, 1 ml de ácido clorhídrico 0,1 N, y agua destilada hasta 5 ml de volumen final. </li><li> - Acetato de solución madre de veronal se obtiene por disolución de 1,15 g de acetato de sodio andydrous y 2,94 g de barbital en 100 ml de agua destilada. </li><li> Solución-Kellenberger contiene 2 ml de acetato de stock veronal, 2,8 ml de ácido clorhídrico 0,1 N, 0,05 g de acetato de uranilo, y 5,1 mlde agua destilada, pH = 6. </li></ol></li><li> Lavar los bloques de pulmón en 0,1 M de cacodilato de sodio tampón, pH = 7,4, 3 veces brevemente. </li><li> Fijar las muestras en el 4% (peso / vol) de formaldehído, 2,5% de glutaraldehído en 0,1 TUBOS tampón, pH 7,2, durante 1 hora, a temperatura ambiente. </li><li> Post-arreglar con un 1% Palade-osmio durante 1 hora en la capilla, en el hielo, en la oscuridad. </li><li> Lavar una vez con tampón Kellenberger. </li><li> Incubar las muestras en tampón de Kellenberger durante 2 horas a toda la noche, a temperatura ambiente. </li><li> Enjuagar una vez en etanol al 50%. </li><li> Deshidratar con serie graduada de etanol, 5 min / cada uno: 70%, 95%, luego 2 x 15 min 100% de etanol. </li><li> Cambiar a óxido de propileno 100%, 2 x 15 min. </li><li> Eliminar el óxido de propileno y añadir una mezcla de óxido de propileno 50% y 50% Epon 812, incubar durante la noche, en una rueda giratoria, a temperatura ambiente. </li><li> La mañana siguiente, retire la mezcla y añadir Epon fresca 812, al menos por 4-5 horas en el volante. </li><li> Utilizando moldes cónicos duplicado, llenosmedio camino con fresca 100% Epon 812, añadir especímenes seleccionados y colocarlos a los bordes. </li><li> Mueva los moldes a la incubadora a 60 ° C y dejarlos curar durante 48-72 horas. </li><li> Cuando se polimeriza, envía los bloques de estar delgado (60-70 nm de espesor) seccionado. </li></ol>

<ol>
	<li>Predescu, S. A., Predescu, D. N., Knezevic, I., Klein, I. K., Malik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin-1s+regulates+the+mitochondrial+apoptotic+pathway+in+endothelial+cells.">Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells.</a> J. Biol. Chem. 282 (282), 17166-17178 (2007).</li><li>Uddin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipids+used+in+non-viral+gene+delivery+systems.">Cationic lipids used in non-viral gene delivery systems.</a> Biotechnology and Molecular Biology Review. 2 (2), 58-67 (2007).</li><li>Barenholz, Y., 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=Influence+of+lipid+composition+on+the+thermotropic+behavior+and+size+distribution+of+mixed+cationic+liposomes.">Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes.</a> J. Colloid Interface Sci. (356), 46-53 (2011).</li><li>Predescu, D. N., Neamu, R., Bardita, C., Wang, M., Predescu, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+caveolae+function+and+upregulation+of+alternative+endocytic+pathways+induced+by+experimental+modulation+of+intersectin-1s+expression+in+mouse+lung+endothelium.">Impaired caveolae function and upregulation of alternative endocytic pathways induced by experimental modulation of intersectin-1s expression in mouse lung endothelium.</a> Biochem. Res. Int.. (2012), 672705 (2012).</li><li>Higuchi, Y., Kawakami, S., Hashida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+in+vivo+delivery+of+siRNAs:+recent+progress.">Strategies for in vivo delivery of siRNAs: recent progress.</a> BioDrugs. 24 (24), 195-205 (2010).</li><li>Caracciolo, G., Amenitsch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposome/DNA+complexes:+from+structure+to+interactions+with+cellular+membranes.">Cationic liposome/DNA complexes: from structure to interactions with cellular membranes.</a> Eur. Biophys. J. , (2012).</li><li>Barnier Quer, C., Elsharkawy, A., Romeijn, S., Kros, A., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposomes+as+adjuvants+for+influenza+hemagglutinin:+more+than+charge+alone.">Cationic liposomes as adjuvants for influenza hemagglutinin: more than charge alone.</a> Eur. J. Pharm. Biopharm. 81 (81), 294-302 (2012).</li><li>Rai, K., 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=Liposomal+delivery+of+MicroRNA-7-expressing+plasmid+overcomes+epidermal+growth+factor+receptor+tyrosine+kinase+inhibitor-resistance+in+lung+cancer+cells.">Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells.</a> Mol. Cancer Ther. (10), 1720-1727 (2011).</li><li>McLean, J. W., 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=Organ-specific+endothelial+cell+uptake+of+cationic+liposome-DNA+complexes+in+mice.">Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice.</a> Am. J. Physiol. (273), H387-H404 (1997).</li><li>Soutschek, J., 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=Therapeutic+silencing+of+an+endogenous+gene+by+systemic+administration+of+modified+siRNAs.">Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.</a> Nature. (432), 173-178 (2004).</li><li>Barenholz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+enhance+bioremediation+of+oil-contaminated+soil.">Liposomes enhance bioremediation of oil-contaminated soil.</a> J. Liposome Res. (13), 1-8 (2003).</li><li>Zamboni, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal,+nanoparticle,+and+conjugated+formulations+of+anticancer+agents.">Liposomal, nanoparticle, and conjugated formulations of anticancer agents.</a> Clin. Cancer Res. (11), 8230-8234 (2005).</li><li>Newman, M. S., Colbern, G. T., Working, P. K., Engbers, C., Amantea, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacokinetics,+tissue+distribution,+and+therapeutic+effectiveness+of+cisplatin+encapsulated+in+long-circulating,+pegylated+liposomes+(SPI-077)+in+tumor-bearing+mice.">Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposomes (SPI-077) in tumor-bearing mice.</a> Cancer Chemother Pharmacol. (43), 1-7 (1999).</li><li>Martino, S., 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=Efficient+siRNA+delivery+by+the+cationic+liposome+DOTAP+in+human+hematopoietic+stem+cells+differentiating+into+dendritic+cells.">Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells.</a> J. Biomed. Biotechnol. (2009), 410260 (2009).</li><li>Zhang, G., 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=A+delivery+system+targeting+bone+formation+surfaces+to+facilitate+RNAi-based+anabolic+therapy.">A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy.</a> Nat. Med. 18 (18), 307-314 (2012).</li><li>Predescu, D., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmalemmal+vesicles+represent+the+large+pore+system+of+continuous+microvascular+endothelium.">Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium.</a> Am. J. Physiol. (265), H725-H733 (1993).</li><li>Koh, T. W., Verstreken, P., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dap160/intersectin+acts+as+a+stabilizing+scaffold+required+for+synaptic+development+and+vesicle+endocytosis.">Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis.</a> Neuron. (43), 193-205 (2004).</li><li>Miyawaki-Shimizu, K., 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=.">.</a> Lung Cell Mol. Physiol. (290), L405-L413 (2006).</li></ol>

Los autores no tienen nada que revelar.

Con base en estudios anteriores publicados por otros 9 y nosotros 1, 18 hemos desarrollado esta metodología para el largo plazo derribar de ITSN-1 in vivo mediante la administración intravenosa repetida (cada 72 horas, durante 24 días consecutivos) de siRNA / liposoma. Este enfoque experimental es eficaz, se puede utilizar de forma segura y en repetidas ocasiones y que se puede extender fácilmente para estudiar la implicación de un gen que codifica cualquier proteína de interés en e...

ARNsi desnudo no puede penetrar la membrana celular, siendo cargado negativamente, y es fácilmente degradado por enzimas en la sangre, tejidos y células. Incluso con recientemente modificaciones estructurales para mejorar la estabilidad, la acumulación de siRNA en el sitio diana después de la administración es extremadamente baja y requiere un vehículo intracelular eficiente 5. Los liposomas catiónicos surgieron como seguras ácidos nucleicos portadores con el potencial de transferir grandes piezas de ADN / ARN en las células mediante la encapsulación y la protección de los ácidos nucleicos a partir de la degradación enzimática 6. También, los liposomas catiónicos espontáneamente interactúan con el ADN / ARN, promoviendo así la transferencia de genes a las células 2. Más recientemente, los liposomas se han aplicado para entregar vacunas y fármacos de bajo peso molecular 7. Entrega liposomal de microARN-7-plásmido que expresa supera factor de crecimiento epidérmico receptor de la tirosina quinasa inhibidor de la resistencia en las células de cáncer de pulmón 8. Cuando el objetivo es laendotelio vascular, la administración intravenosa es esencial, ya que los complejos son poco probable que cruzar la barrera endotelial y extravasación en el intersticio 9. En comparación a otros órganos, ECs de la microvasculatura del pulmón tener el mayor absorción y ávidamente internalizar liposomas catiónicos y complejos de ADN / ARN, seguido por los ganglios linfáticos y los parches de Peyer 9. La entrega por vía intravenosa en modelos de roedores de ARNsi a través de inyecciones en la vena retro-orbital o de la cola ha sido probado ser inofensivas incluso en altas concentraciones, tales como 50 mg / kg 10. En la literatura publicada la composición de los liposomas catiónicos es diferente, basada en la formulación de lípidos y su relación equimolar 11, 12. Hay una amplia gama de aplicaciones potenciales utilizando liposomas catiónicos para la entrega de genes in vivo, ya sea dirigiéndose down-regulation/over-expression de las proteínas, la entrega de vacunas o terapias anti-tumorales 13-15. Es importante recordares que la eficiencia de ADN / ARN-interacción membrana celular está regulada por el acoplamiento de forma entre los complejos generados y lípidos de membrana. Esto sugiere que la adaptación de la composición de los lípidos de los perfiles de la membrana celular específicos puede ser beneficioso y el resultado en altas tasas de transfección en un tipo de célula particular 6.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Dioctadecyl Ammonium Bromide (DOAB)</td>
 <td>Sigma-Aldrich</td>
 <td>D2779</td>
 <td></td>
 </tr>
 <tr>
 <td>LiposoFast stabilizer (mini-extruder)</td>
 <td>Avestin Inc.</td>
 <td>LF-STB</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate membrane, 19 mm diameter, 50 nm pore</td>
 <td>Avestin Inc.</td>
 <td>LFM-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Cholesterol</td>
 <td>Sigma-Aldrich</td>
 <td>C-8667</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Mallinckrodt Chemicals</td>
 <td>4432-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's balanced salts</td>
 <td>Sigma-Aldrich</td>
 <td>H1387</td>
 <td></td>
 </tr>
 <tr>
 <td>Round-bottom flask</td>
 <td>Fisher Scientific</td>
 <td>FHB-275-030X</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse siRNAITSN - on target </td>
 <td>Dharmacon/ThermoScientific</td>
 <td>J-046912-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pump</td>
 <td>Ismatec</td>
 <td>REGLO-CDF digital with RHOO pump head</td>
 <td></td>
 </tr>
 <tr>
 <td>Ventilator</td>
 <td>Hugo Sachs Electronik</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>Barbital</td>
 <td>Sigma-Aldrich</td>
 <td>B-0500</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>EM Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>Epon812</td>
 <td>EM Sciences</td>
 <td>Discontinued by the manufacturer</td>
 <td></td>
 </tr>
 <tr>
 <td>Propylene oxide</td>
 <td>EM Sciences</td>
 <td>20400</td>
 <td></td>
 </tr>
 <tr>
 <td>Embedding molds</td>
 <td>EM Sciences</td>
 <td>69923-05</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>EM Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 <tr>
 <td>8 nm gold-albumin tracer</td>
 <td></td>
 <td>Prepared in the laboratory as in16</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Pyrex, 100 ml</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Modified siRNA, in vivo</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Part of IDEX corp.</td>
 </tr>
 <tr>
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 <td>D-79232 March, Germany</td>
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long-term silencing of intersectin-1s in mouse lungs by repeated delivery of a specific sirna via cationic liposomes. evaluation of knockdown effects by electron microscopy

Estudios anteriores mostraron que desmontables de ITSN-1s (KD ITSN), una proteína implicada en la regulación endocítica permeabilidad vascular pulmonar y células endoteliales (EC) la supervivencia, la muerte celular apoptótica inducida, un obstáculo importante en el desarrollo de un sistema de cultivo celular con prolongada ITSN-1s inhibición 1. Utilizando liposomas catiónicos como vehículos, exploramos el silenciamiento de genes ITSN-1 en los pulmones de ratones mediante la administración sistémica de siRNA dirigidos ITSN-1 gen (ITSN siRNA). Los liposomas catiónicos ofrecen varias ventajas para la salida del siRNA: caja fuerte con dosificación repetida, no inmunogénico, no tóxico, y fácil de producir 2. Los liposomas rendimiento y la actividad biológica dependen de su tamaño, carga, composición de lípidos, la estabilidad, la dosis y la vía de administración 3 Aquí, eficiente y ITSN KD específica en los pulmones del ratón se ha obtenido mediante una combinación bromuro de amonio y dimetil dioctadecil colesterol. La administración intravenosade ITSN siRNA / complejos de liposomas catiónicos transitoriamente derribaron proteína ITSN-1 y mRNA en los pulmones de ratones en el día 3, que se recuperó después de 3 días adicionales. Tomando ventaja de los liposomas catiónicos como un portador seguro repetible, el estudio extendió durante 24 días. Por lo tanto, el tratamiento retro-orbital con complejos recién generados se administró cada 3 días, induciendo ITSN KD sostenida a lo largo del estudio 4. Tejidos de ratón recogidos en varios puntos de tiempo post-siRNA ITSN fueron sometidos a análisis de microscopía electrónica (EM) para evaluar los efectos de la crónica KD ITSN, en el endotelio pulmonar. De alta resolución de imágenes EM nos permitió evaluar los cambios morfológicos causados ​​por KDITSN en el lecho vascular pulmonar (es decir, la interrupción de la barrera endotelial, disminución del número de caveolae y la regulación positiva de las vías de transporte alternativos), las características no detectables por microscopía de luz. En general estos resultados establecieron un importantetante papel de ITSN-1 en la función de EC y la homeostasis de pulmón, mientras que ilustra la eficacia de la entrega de siRNA-liposomas in vivo.

Los niveles de proteína ITSN-1s y el ARNm se controlaron a varios puntos de tiempo después de siRNA entrega ITSN por Western blot y PCR convencional y cuantitativo como en 4. Los niveles de proteína ITSN-1s y ARNm en siRNA ITSN pulmones tratados con ratón fueron aproximadamente 75% más baja por referencia a los controles durante la caída continua de ITSN-1s durante 21 días. Sin ITSN-1s, dynamin-2, una importante interacción socio de jugador ITSN-1s y esencial en el desprendimient...

Watch this Scientific Journal Video about Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Elect

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Inyecciones retro-orbitales repetidas de complejos catiónicos liposomes/siRNA/ITSN-1s en ratones, cada 72 horas durante 24 días, eficiente entregar el dúplex siARN de la microvasculatura pulmonar del ratón la reducción de ARNm ITSN-1s y expresión de la proteína en un 75%. Esta técnica es altamente reproducible en un modelo animal, no tiene efectos adversos y evita accidentes mortales.

Silenciar a largo plazo de Intersectin-1 en los pulmones de ratones por entrega repetida de un siRNA específico a través de liposomas catiónicos. Evaluación de Efectos desmontables de Microscopía Electrónica

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

long-term-silencing-intersectin-1s-mouse-lungs-repeated-delivery

Research

JoVE Journal

Biology

10.6K vistas.  Rush University. Inyecciones retro-orbitales repetidas de complejos catiónicos liposomes/siRNA/ITSN-1s en ratones, cada 72 horas durante 24 días, eficiente entregar el dúplex siARN de la microvasculatura pulmonar del ratón la reducción de ARNm ITSN-1s y expresión de la proteína en un 75%. Esta técnica es altamente reproducible en un modelo animal, no tiene efectos adversos y evita accidentes mortales.

Video: Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicapa de montaje para microscopía de luz Hoja de largo plazo del pez cebra

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

La visualización de la ATP sintasa dímeros en mitocondrias por Electron Cryo-tomografía

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

En Vivo transfección siRNA y Gene Derribo en la médula espinal a través de Rapid no invasiva lumbar intratecal inyecciones en ratones

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Cryo-Microscopía Electrónica de Preparación de Muestras por medio de un haz de iones focalizados

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

Correlativa confocal y microscopía electrónica de 3D de una célula sensorial específica

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Evaluación del huso meiótico en ovocitos de ratón por silenciamiento siRNA mediada

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparación de la muestra y proyección de imagen de exosomas por microscopía electrónica de transmisión

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...