Este trabajo fue apoyado por las becas de la Fundación de Ciencias naturales nacionales de China (31201086, 31470759 y 31671219) y la Fundación de Ciencias naturales de Shanghai (12ZR1436900).

ratones C57BL/6 se crían en condiciones libres de patógenos específicas. Todos los métodos experimentales de animales están en conformidad con las directrices aprobadas por el cuidado Animal y Comité de ética en la Universidad de medicina militar de segunda.
 1. disección de Postnatal desarrollo de glándula pituitaria <ol><li>realizar anestesia: poner ratones neonatales (P0 - P5) en hielo picado para inducir hipotermia. Para los ratones mayores de 5 días de edad, inyectar 5% uretano (30 μl/g de peso corporal) por vía intraperitoneal para inducir anestesia. Evaluar las respuestas para toe cola pinches. Proceder sólo después de que el ratón no responde a los estímulos nocivos.</li>
	<li>Garantizar el ratón en la posición supina (acostado boca arriba con la cara hacia arriba) con una cinta suavemente las patas delanteras y fursuit a una superficie de trabajo que es capaz de ser fijado (es decir, espuma de poliestireno) dentro de una campana química.</li>
	<li>Realizar la perfusión transcardial.
	<ol><li>Corte la caja torácica de ratón con tijeras quirúrgicas para exponer el corazón (y otros órganos torácicos).</li>
		<li>Garantizar el corazón con el fórceps romos e Inserte una aguja 26G (0,45 mm) en el ventrículo izquierdo. La aguja está conectada a una jeringa de 10 mL llenada con solución salina heparinizada tamponada de fosfato (PBS; complementado con nitrito de sodio 5 mg/mL y 10 U/mL heparina, pH 7,4, calentar a 37 ° C antes de usar). Inmediatamente corte el atrio derecho con unas tijeras finas y comienzan a inundar el cuerpo con el PBS caliente hasta que el líquido que sale de la aurícula derecha es claro. 
		Nota: Aproximadamente 5-10 mL de PBS es necesario para los ratones neonatales y 10-20 mL de PBS para los ratones adultos.</li>
		<li>Mantener la aguja en el lugar y cambiar a otra jeringa llenado con paraformaldehído al 4% (PFA, pH 7,4). Perfusión de 10 mL y 20 mL de fijador para neonatal y P21 ratón, respectivamente. 
		PRECAUCIÓN: El PFA es tóxico. Puede causar irritaciones respiratorias y la piel y daños oculares. Guantes y trabajar bajo una campana química.</li>
	</ol></li> <li>Decapitar el ratón fijo de PFA y cortar el hueso del cráneo abierto con tijeras.
	<ol><li>Levante suavemente el cerebelo de la base del cráneo con pinzas pequeñas. En el primer signo de la silla turca, parada de elevación pero mantenga el cerebelo, corta el tallo pituitario y las fibras nerviosas de conexión a la base del cerebro con unas tijeras finas. Este paso es vital para asegurar que la hipófisis no se levanta, con el cerebro.</li>
		<li>Luego seguir levantando el cerebro y retire todo el cerebro para exponer completamente la glándula pituitaria. Cortar la región sella todo incluyendo la glándula pituitaria, laterales de los nervios de trigeminal y debajo del hueso esfenoides con tijeras (aproximadamente 3 x 5 mm 2).</li>
	</ol></li> <li>Poner el tejido disecado en un plato de 35 mm o un pocillo de una placa de 6 pozos que contienen 2 mL frío 4% PFA (pH 7.4). Fijar el tejido en 4 ° C por 40 min para P0 - P7 pituitaries, 1 h para P14 pituitaries, 1,5 h de P21 - P28 pituitaries y 3 h para adultos pituitaries.</li>
	<li>Mayor disociación del pituitary <ol><li>Lave el tejido fijo con 10 mL de PBS, 5 cambios, 15 minutos cada uno. La glándula pituitaria neonatal junto con los tejidos circundantes y por debajo del hueso esfenoidal pueden procesarse directamente a la deshidratación. Sin embargo, se debe disociar glándulas pituitarias de los ratones mayores de 5 días más un microscopio estéreo.</li>
		<li>Poner el tejido fijo en un plato de 35 mm que contiene 1 mL de PBS. P5 - P14 pituitaries, quitar las membranas conectivas entre los nervios y el hueso con finas pinzas y tijeras y aislar cuidadosamente la hipófisis junto con los nervios de trigeminal lateral como un todo, pero dejando el turcica del sella. Los aislados de la glándula y los nervios están conectados por membranas de tejido conectivas con una &#34; H &#34;-como el aspecto ( figura 1B).</li>
		<li>Para P21 y adulto pituitaries, quitar los nervios y membranas de tejido conectivas alrededor de la glándula pituitaria y sin la glándula de los tejidos circundantes. Envolver el tejido hipofisario con papel de seda para limpiar lentes y ponerlo en un cassette inclusión. 
		Nota: Envolver la hipófisis pequeña con papel de seda para limpiar lentes ayuda a prevenir cualquier posible pérdida de especímenes y preservar la integridad del tejido en el siguiente procedimiento de inclusión en parafina. No puede ser necesario envolver la hipófisis si se procesa en una pequeña botella en el procedimiento de inclusión de cryo.</li>
	</ol></li></ol> 2. Incorporación de parafina secciones <ol><li>deshidratar los especímenes en la encajadura casetes con soluciones de etanol 50%, 65%, 75% y 95% por 15 min. Posteriormente deshidratar con 3 cambios de etanol puro, 3 min para P0 - P4 glándulas, 4 min para P5 - P14 glándulas y 5 min para P21 y glándulas mayores. Claro con xileno, 3 cambios, 3 min para P0 - P4 glándulas, 4 min para P5 - P14 glándulas y 5 min para P21 y glándulas mayores. Infiltrarse con cera de parafina fundida, 3 cambios, 7 min de P0 - P4 glándulas, y 8 min dos veces seguida de 6 min una vez para P5 y glándulas mayores. 
	Nota: Los parámetros óptimos para el proceso de tejido pueden ser ajustados empíricamente. Para obtener secciones de alta calidad, debe evitarse la deshidratación insuficiencia o excedente. Lo mismo vale para limpiar el tejido con xileno.</li>
	<li>Orientación cuando incrustar <ol><li>en un sistema de la consola, para inclusión tisular retirar la muestra del cassette y lo coloca en un molde base lleno hasta la mitad con cera de parafina fundida. La orientación correcta de la glándula pituitaria incrustación es esencial para satisfacer las secciones coronales.</li>
		<li>Para P21 y adulto las glándulas pituitaria, posición de la glándula pituitaria con su eje corto perpendicular a la superficie inferior de un molde de base (el plano de sección). Utilice los huesos esfenoides y los nervios de trigeminal como puntos de referencia anatómicos para P0 - P4 y P5 - P14 pituitaries, respectivamente. Orientar las muestras con sus nervios de trigeminal o lámina transversal de los huesos esfenoides perpendiculares a la superficie inferior de un molde base.</li>
		<li>Suavemente Sujete el tejido en la posición deseada con el fórceps fino calentado hasta que la cera se vuelve semi sólida en una placa de enfriamiento. Completar el molde con cera de parafina fundida. Permitir que el bloque de parafina se enfríe hasta que la cera se solidifica completamente. 
		Nota: La glándula pituitaria en el día embrionario 18.5 (E18.5) puede ser tratada de la misma manera como la hipófisis neonatal. Pero los pituitaries (Rathke &#39; bolsas s) menores E16.5 debe ser integrado con toda la cabeza y orientado con el eje sagital (de boca al occiput) perpendicular a la superficie inferior de un molde base.</li>
	</ol></li> <li>Enfriar el bloque de parafina a-20 ° C durante 10-20 minutos y luego córtela en finas rodajas (4 μm de grosor se prefiere) con un micrótomo. Para obtener secciones coronales satisfactorias, ajustar la posición de bloques de parafina durante el seccionamiento. Montar los cortes de tejido en portaobjetos recubiertos polylysine para evitar desprendimiento de láminas en los siguientes procedimientos. 
	Nota: Como alternativa, muestras pueden ser deshidratadas en 15%, 30% (P/V) soluciones de sacarosa (en PBS) a 4 ° C hasta que hunda hasta el fondo, incrustado en cryo-incrustar compuesto utilizando el mismo método de orientación y rápidamente congelada en hielo seco. Luego cortar los bloques de tejido congelado de 8-10 μm con un criostato. La morfología de cryo-secciones, sin embargo, es a menudo inferior a parafina secciones.</li>
</ol> 3. H &#38; E coloración y etiquetado inmunofluorescencia <ol><li>caliente las diapositivas en un calor bloquean a 55 ° C y dewax con xileno, 2 cambios, 4 min. Deshidratan las muestras con etanol al 95% dos veces y el etanol del 75% dos veces, 3 minutos cada uno. Lavar los portaobjetos con agua destilada durante 5 min en un agitador.</li>
	<li>De H &#38; E coloración, mancha las secciones de solución de hematoxilina de alumbre durante 6 min., enjuagar en runnagua corriente por 2 min, diferenciar con 0.2% amoniaco agua para 45 s y volver a enjuagar en agua durante 2 minutos. Luego deshidratar las secciones con etanol al 95% por 1 min y tinción con eosina durante 2 minutos secuencialmente deshidratan, claro y monten.</li>
	<li>Etiquetado de inmunofluorescencia <ol><li>trata las secciones con metanol que contenga 3% H 2 O 2 y 0.01% NaN 3 por 20 min a temperatura ambiente para inactivar peroxidasas endógenas. Recuperar antígenos hirviendo cortes en el tampón de recuperación (1 mM EDTA y 1 mM Tris-HCl, pH 7,4) en una olla a presión durante 2 minutos incubar las secciones con solución amortiguadora de bloqueo (5% de suero de cabra en PBS) durante 30 min a 37 ° C.</li>
		<li>Incubar secciones con anticuerpos hormona del crecimiento (GH) (1:2, 000) o anticuerpos de la proteína ácida fibrilosa anti-glial (GFAP) conejo (1:2, 000) diluidas en solución amortiguadora de bloqueo durante la noche a 4 ° C.</li>
		<li>Incubar secciones con anticuerpo secundario (cabra anti-conejo IgG-HRP, 1:300 en solución amortiguadora de bloqueo) durante 35 min a 37 ° C.</li>
		<li>Amplificar las señales usando Biotinyl Tyramide (1:50 en solución salina tamponada con Tris que contenga 0.3% H 2 O 2) por 15 min a temperatura ambiente y visualizar con estreptavidina Alexa594 o 488 (1:300 en el bloqueo de almacenador intermediario, 30 min, 37 º C).</li>
		<li>Contratinción los núcleos con DAPI (50 mg/L de PBS) durante 5 minutos a temperatura ambiente. 
		Nota: Puede no ser necesario amplificar la señal mediante el sistema de amplificación de señal de Tyramide (TSA). Alternativamente, un anticuerpo secundario conjugado fluorescencia puede utilizarse para visualizar la señal de.</li>
	</ol></li> <li>Adquirir imágenes mediante un microscopio de fluorescencia equipado con DAPI, Texas Red y sistemas de filtro FITC, × 4/0,13 × 40/0.75 objetivos y cámara de 5.0 megapíxeles. Uso la 360, 488 y 594 nm láser las longitudes de onda de imágenes DAPI, Alexa 488 - y las secciones manchadas de Alexa 594, respectivamente. Optimizar la energía del laser (0.8 fue utilizado generalmente) y tiempo de exposición a los niveles deseados para cada canal. Capturar la imagen bajo el control del software de adquisición de imagen y superponer las imágenes de fluorescencia posteriormente utilizando software de procesamiento de imagen.</li>
</ol>

<ol>
	<li>Fauquier, T., Lacampagne, A., Travo, P., Bauer, K., Mollard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+face+of+the+anterior+pituitary.">Hidden face of the anterior pituitary.</a> Trends Endocrinol Metab. 13 (7), 304-309 (2002).</li><li>Haedo, M. R., 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=Regulation+of+pituitary+function+by+cytokines.">Regulation of pituitary function by cytokines.</a> Horm Res. 72 (5), 266-274 (2009).</li><li>Zhu, X., Gleiberman, A. S., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+pituitary+development:+signaling+and+transcriptional+networks.">Molecular physiology of pituitary development: signaling and transcriptional networks.</a> Physiol Rev. 87 (3), 933-963 (2007).</li><li>Bancalari, R. E., Gregory, L. C., McCabe, M. J., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+gland+development:+an+update.">Pituitary gland development: an update.</a> Endocr Dev. 23, 1-15 (2012).</li><li>Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+pituitary+gland+development+in+human+and+mouse.">Genetic regulation of pituitary gland development in human and mouse.</a> Endocr Rev. 30 (7), 790-829 (2009).</li><li>Peker, S., Kurtkaya-Yapicier, O., Kilic, T., Pamir, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+anatomy+of+the+lateral+walls+of+the+pituitary+fossa.">Microsurgical anatomy of the lateral walls of the pituitary fossa.</a> Acta Neurochir (Wien). 147 (6), 641-648 (2005).</li><li>Wolpert, S. M., Molitch, M. E., Goldman, J. A., Wood, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size,+shape,+and+appearance+of+the+normal+female+pituitary+gland.">Size, shape, and appearance of the normal female pituitary gland.</a> AJR Am J Roentgenol. 143 (2), 377-381 (1984).</li><li>Carbajo-Perez, E., Watanabe, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+proliferation+in+the+anterior+pituitary+of+the+rat+during+the+postnatal+period.">Cellular proliferation in the anterior pituitary of the rat during the postnatal period.</a> Cell Tissue Res. 261 (2), 333-338 (1990).</li><li>Nantie, L. B., Himes, A. D., Getz, D. R., Raetzman, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+postnatal+pituitary+expansion:+proliferation,+progenitors,+and+cell+specification.">Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.</a> Mol Endocrinol. 28 (5), 731-744 (2014).</li><li>Tzou, S. C., Landek-Salgado, M. A., Kimura, H., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+pituitary+immunogen+for+the+induction+of+experimental+autoimmune+hypophysitis.">Preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis.</a> J Vis Exp. (46), (2010).</li><li>Budry, L., 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=Related+pituitary+cell+lineages+develop+into+interdigitated+3D+cell+networks.">Related pituitary cell lineages develop into interdigitated 3D cell networks.</a> Proc Natl Acad Sci U S A. 108 (30), 12515-12520 (2011).</li><li>Wilson, D. B., Wyatt, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+the+pituitary+gland+in+neonatal+little+(lit)+mutant+mice.">Histopathology of the pituitary gland in neonatal little (lit) mutant mice.</a> Histol Histopathol. 7 (3), 451-455 (1992).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12 (2), 141-149 (1984).</li><li>Cao, D., 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=ZBTB20+is+required+for+anterior+pituitary+development+and+lactotrope+specification.">ZBTB20 is required for anterior pituitary development and lactotrope specification.</a> Nat Commun. 7, 11121 (2016).</li></ol>

Los autores no tienen nada que revelar.

Para el desarrollo de pituitaries murinos, ha sido técnicamente difícil de obtener secciones coronales adecuadas debido a su diminutas y frágil características y características anatómicas únicas6,8. Algunos grupos de investigación así eligieron secciones sagital para analizar la morfología embrionaria y neonatal pituitaria11,12. Aunque la sección sagital de la hipófisis también es capaz de m...

La glándula pituitaria o hipófisis es un importante órgano endocrino que secreta hormonas esenciales para la homeostasis1,2. Anatómicamente, la glándula pituitaria es una estructura de '' dos en uno '' que consiste en la neurohipófisis y la adenohipófisis. Estas piezas tienen función y diferente origen embrionario muy diferentemente. La neurohipófisis deriva del ectodermo neural y segrega oxitocina y hormona antidiurética. La adenohipófisis se origina de la bolsa de Rathke y es responsable de la liberación de hormonas, incluyendo hormona del crecimiento, prolactina, tirotropina, hormona folículo estimulante, hormona luteinizante, hormona adrenocorticotrópica, y hormona estimulante de melanocitos3,4,5.
La glándula pituitaria se basa en la superficie dorsal del hueso esfenoides (silla turca) del cráneo de ratón y se une a la planta del cerebro por un tallo frágil. Lateralmente está rodeado por los nervios trigémino y anterior por el quiasma óptico6,7. La glándula tiene una forma oval alargada con su eje perpendicular a la de la cabeza. En su superficie dorsal, la neurohipófisis y la adenohipófisis pueden fácilmente ser demarcadas, con el anterior ocupando la región medial dorsal y extender este último lateral y ventralmente. Durante el desarrollo postnatal, el tamaño de la hipófisis aumenta rápidamente en el primer mes después de nacimiento8,9. Sin embargo, la hipófisis de ratón todavía es muy pequeña en tamaño con un peso promedio de 1,9 mg y un diámetro de eje largo de ~ 3 mm en adultos10, un diámetro de eje largo de 2-2.5 mm en el día postnatal 21 (P21) y sólo 1-1.5 mm en P0.
Una sección coronal es preferida para mostrar en una sola rebanada de la hipófisis de ratón la adenohipófisis y neurohipófisis. Sin embargo, algunas habilidades técnicas se requieren para obtener satisfacción de secciones coronales de glándulas pituitarias de desarrollar ratones debido a su tamaño excepcionalmente pequeño y la distinta anatomía. En este artículo de vídeo, nos muestran cómo diseccionar glándulas pituitarias de ratón y preparar las secciones coronales pituitarias en diversas etapas del desarrollo.

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors-straight</td>
			<td>JinZhong</td>
			<td>J21010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine scissors-strainght</td>
			<td>JinZhong</td>
			<td>WA1010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>JinZhong</td>
			<td>JD1020</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>RS-5015</td>
			<td>for isolation of the pituitary</td>
		</tr>
		<tr>
			<td>26G (0.45mm) needle</td>
			<td>HongDa</td>
			<td></td>
			<td>for transcardial perfusion</td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>BD</td>
			<td>300841</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>BD</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Lens cleaning paper</td>
			<td>ShuangQuan</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Anatomical microscope</td>
			<td>OLYMPUS</td>
			<td>SZX-ILLB2-200</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td>Thermo Fisher</td>
			<td>22-272423</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Tissue embedding console system</td>
			<td>KEDEE</td>
			<td>KD-BM11</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher</td>
			<td>HM315R</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Superfrost-Plus slides</td>
			<td>Thermo Fisher</td>
			<td>22-037-246</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Thermo Fisher</td>
			<td>12-543</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>BH2-RFCA</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>BBI</td>
			<td>EB0448</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4.12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection</td>
			<td>SPH</td>
			<td>H31022051</td>
			<td>for perfusion saline</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SCR</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Thermo Fisher</td>
			<td>8330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>H9627</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>rabbit anti growth hormone (GH)</td>
			<td>National Hormone</td>
			<td></td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>antibody</td>
			<td>Pituitary Program</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse GFAP antibody</td>
			<td>Sigma</td>
			<td>G9269</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, HRP</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>TSA system</td>
			<td>NEN Life Science Products</td>
			<td>NEL700</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594</td>
			<td>Thermo Fisher</td>
			<td>S32356</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-FITC Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A11090</td>
			<td>for immunostaining</td>
		</tr>
	</tbody>
</table>

dissection and coronal slice preparation of developing mouse pituitary gland

La glándula pituitaria o hipófisis es un importante órgano endocrino que secreta hormonas esenciales para la homeostasis. Consta de dos glándulas con funciones y orígenes embrionarios separados, la neurohipófisis y la adenohipófisis. La hipófisis de ratón en desarrollo es pequeño y delicado con una forma oval alargada. Una sección coronal es preferida para mostrar la adenohipófisis y la neurohipófisis en una sola rebanada de la hipófisis de ratón.
El objetivo de este protocolo es lograr la correcta secciones coronales pituitarias con arquitecturas de tejido preservado de desarrollar ratones. En este protocolo, se describe en detalle cómo diseccionar y glándulas pituitarias correctamente de desarrollar ratones. En primer lugar, ratones son fijados por perfusión de transcardial del formaldehído antes de la disección. Luego se aplican tres diferentes técnicas de disección para obtener las glándulas pituitaria intactas dependiendo de la edad de los ratones. Para ratones fetales a partir de 17.5-18.5 días embrionarios (E) y los recién nacidos hasta 4 días, las regiones de sella todo incluyendo el hueso esfenoides, glándulas y nervios de trigeminal se disecan. Para cachorros edad postnatales días (P) 5-14, las glándulas pituitaria conectados con los nervios de trigeminal son disecados en su conjunto. Para ratones de 3 semanas de edad, las glándulas pituitarias se disecan con cuidado gratis de los tejidos circundantes. También nos muestra cómo incrustar las glándulas pituitarias en una orientación adecuada mediante el uso de los tejidos circundantes como puntos de referencia para obtener satisfacción de secciones coronales. Estos métodos son útiles en el análisis de características histológicas y del desarrollo de las glándulas de la hipófisis en el desarrollo de ratones.

Este protocolo presenta un método para disecar glándulas pituitarias de desarrollar ratones. Para el ratón neonatal, las regiones de sella todo que contiene la glándula pituitaria, los nervios de trigeminal y la parte inferior se diseca hueso esfenoides de la base del cráneo. La hipófisis pequeña y delicada permaneció intacta durante el proceso (figura 1A). Para los ratones mayores de 5 días, las glándulas pituitaria conectadas a los nervios de trig...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

Disección y preparación del segmento Coronal de desarrollo de hipófisis de ratón

Presentamos un protocolo para diseccionar las glándulas pituitaria y preparar pituitarias secciones coronales de desarrollar ratones.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

dissection-coronal-slice-preparation-developing-mouse-pituitary

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

17.1K vistas.  Second Military Medical University. Presentamos un protocolo para diseccionar las glándulas pituitaria y preparar pituitarias secciones coronales de desarrollar ratones.

Video: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero electroporación y organotípicos rebanada Cultura de ratón Hipocampo Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Investigación

Biología del Desarrollo

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Métodos para examinar los nódulos linfáticos y en los hemocitos<em&gt; Drosophila</em&gt; Las larvas

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Vivir Imagen confocal de desarrollo de Arabidopsis Flores

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

La disección de las larvas de pez cebra de tejidos gonadales

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Disección y cultivo de riñón embrionario de ratón

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Disección y la coloración de los ovarios Pupal de Drosophila

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

La detección de Immunohistochemical de la 5-METILCITOSINA y 5-hidroximetilcitosina en desarrollo y en la Retina de ratón Postmitotic

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

Evaluación de parámetros de neuroplasticidad ultraestructurales después en la transducción de útero de cerebro de ratón en desarrollo y a la médula espinal

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Preparación de tejidos e inmunomanchación de tejidos craneofaciales de ratón y hueso no descalcificado

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Disección, cultivo y análisis de células de cresta neural craneal primarias de ratón para el estudio de la delaminación y migración de células de cresta neuronal

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...