Este trabajo fue apoyado por el NIH subvenciones R21MH082360 (BGC) y R01HD056315 (MNR), así como la Universidad de Georgia.

<ol>
	<li>Divjak, M., Glare, E. M., Walters, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+non-radioactive+in+situ+hybridization+in+human+airway+tissues:+use+of+PCR-generated+templates+for+synthesis+of+probes+and+an+antibody+sandwich+technique+for+detection+of+hybridization.">Improvement of non-radioactive in situ hybridization in human airway tissues: use of PCR-generated templates for synthesis of probes and an antibody sandwich technique for detection of hybridization.</a> J. Histochem. Cytochem. 50, 541-548 (2002).</li><li>Wilkinson, D. G., Nieto, 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=Detection+of+messenger+RNA+by+in+situ+hybridization+to+tissue+sections+and+whole+mounts.">Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.</a> Methods Enzymol. 225, 361-373 (1993).</li><li>Logel, J., Dill, D., Leonard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cRNA+probes+from+PCR-generated+DNA.">Synthesis of cRNA probes from PCR-generated DNA.</a> Biotechniques. 13, 604-610 (1992).</li><li>Maddox, D. M., Condie, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+expression+of+a+glutamate+decarboxylase+gene+in+multiple+non-neural+tissues+during+mouse+development.">Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development.</a> BMC Dev Biol. 1, 1-1 (2001).</li><li>Conlon, R. A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+retinoic+acid+rapidly+induces+anterior+ectopic+expression+of+murine+Hox-2+genes+in+vivo.">Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo.</a> Development. 116, 357-368 (1992).</li><li>Krauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+pax[zf-a]:+a+paired+box-containing+gene+expressed+in+the+neural+tube.">Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube.</a> EMBO J. 10, 3609-3619 (1991).</li><li>Hemmati-Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+specific+mRNAs+in+Xenopus+embryos+by+whole-mount+in+situ+hybridization.">Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.</a> Development. 110, 325-330 (1990).</li><li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+an+improved+whole-mount+method+for+Xenopus+embryos.">In situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Methods Cell Biol. 36, 685-695 (1991).</li><li>Herrmann, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+the+Brachyury+gene+in+whole-mount+TWis/TWis+mutant+embryos.">Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.</a> Development. 113, 913-917 (1991).</li><li>Conlon, R. A., Herrmann, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+messenger+RNA+by+in+situ+hybridization+to+postimplantation+embryo+whole+mounts.">Detection of messenger RNA by in situ hybridization to postimplantation embryo whole mounts.</a> Methods Enzymol. 225, 373-383 (1993).</li><li>Parr, B. A., Shea, M. J., Vassileva, G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Wnt+genes+exhibit+discrete+domains+of+expression+in+the+early+embryonic+CNS+and+limb+buds.">Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.</a> Development. 119, 247-261 (1993).</li><li>Rosen, B., Beddington, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridization+in+the+mouse+embryo:+gene+expression+in+three+dimensions.">Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.</a> Trends Genet. 9, 162-167 (1993).</li><li>Quiring, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+expression+screening+by+automated+whole-mount+in+situ+hybridization.">Large-scale expression screening by automated whole-mount in situ hybridization.</a> Mech. Dev. 121, 971-976 (2004).</li><li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev. Biol. 231, 63-76 (2001).</li><li>Neidhardt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screen+for+genes+controlling+mammalian+embryogenesis,+using+high-throughput+gene+expression+analysis+in+mouse+embryos.">Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.</a> Mech. Dev. 98, 77-94 (2000).</li><li>Gordon, J., Bennett, A. R., Blackburn, C. C., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gcm2+and+Foxn1+mark+early+parathyroid-+and+thymus-specific+domains+in+the+developing+third+pharyngeal.">Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal.</a> 103, 141-143 (2001).</li></ol>

No hay conflictos de interés declarado.

Los métodos descritos en este protocolo ha sido adaptado de una serie de fuentes diferentes y optimizado para montaje en toda E8.5-E11.5 embriones día de vida del ratón. Los métodos para montar toda la hibridación in situ de embriones de los vertebrados aparecieron por primera vez a principios de 1990 2,5-12. Este protocolo fue adaptado sobre todo de los métodos desarrollados para embriones de Xenopus 7,8 y 2,11 ratón. En nuestro protocolo de una gran cantidad de...

<table border="1"><tbody><tr><th> Nombre </th><th> Tipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Digoxigenina-11-uridina-5&#39;-trifosfato </td><td> Reactivo </td><td> Roche </td><td> 11209256910 </td><td></td></tr><tr><td> Ribonucleósido Set trifosfato </td><td> Reactivo </td><td> Roche </td><td> 11277057001 </td><td></td></tr><tr><td> Columnas Un paseo rápido por la purificación de ARN radiomarcado </td><td> Suministro </td><td> Roche </td><td> 11274015001 </td><td></td></tr><tr><td> T7 RNA polimerasa </td><td> Reactivo </td><td> Roche </td><td> 10881767001 </td><td></td></tr><tr><td> SP6 RNA polimerasa </td><td> Reactivo </td><td> Roche </td><td> 10810274001 </td><td></td></tr><tr><td> T3 ARN polimerasa </td><td> Reactivo </td><td> Roche </td><td> 11031163001 </td><td></td></tr><tr><td> CTP, [α-32P] - 800Ci/mmol 10mCi/ml, 250 Ci </td><td> Reactivo </td><td> Perkin Elmer </td><td> BLU008X250UC </td><td></td></tr><tr><td> DNasa I recombinante, libre de RNasa </td><td> Reactivo </td><td> Roche </td><td> 4716728001 </td><td></td></tr><tr><td> Urea, cristales incoloros a blancos o polvo cristalino </td><td> Reactivo </td><td> Pescador </td><td> BP169-500 </td><td></td></tr><tr><td> Gel de tampón de carga </td><td> Reactivo </td><td> Ambion </td><td> AM8547 </td><td></td></tr><tr><td> Hybond-N +, Amersham </td><td> Suministro </td><td> GE Healthcare </td><td> RPN82B </td><td></td></tr><tr><td> UV Stratalinker 2400 </td><td> Equipo </td><td> Stratagene </td><td></td><td></td></tr><tr><td> Dietil pirocarbonato </td><td> Reactivo </td><td> Sigma </td><td> D5758-100ML </td><td></td></tr><tr><td> Heparina sódica </td><td> Reactivo </td><td> Acros </td><td> 41121-0010 </td><td></td></tr><tr><td> peróxido de hidrógeno al 30% en agua </td><td> Reactivo </td><td> Fisher Scientific </td><td> BP2633-500 </td><td></td></tr><tr><td> Glutaraldehído, el 8% de grado EM </td><td> Reactivo </td><td> Polysciences </td><td> 0 / 710 </td><td> Utilizar con precaución de acuerdo con las instrucciones del fabricante de </td></tr><tr><td> El bloqueo de los reactivos </td><td> Reactivo </td><td> Roche </td><td> 11096176001 </td><td> Convertirlo en un 5% de acciones y almacenar a -20 ° C </td></tr><tr><td> Proteinasa K </td><td> Reactivo </td><td> Roche </td><td> 3115852001 </td><td></td></tr><tr><td> RNasa A </td><td> Reactivo </td><td> Roche </td><td> 10109142001 </td><td> Hacer como 10 mg / ml y almacenar a -20 ° C. </td></tr><tr><td> RNasa T1 </td><td> Reactivo </td><td> Roche </td><td> 10109193001 </td><td></td></tr><tr><td> Ultrapura formamida </td><td> Reactivo </td><td> Invitrogen </td><td> 15515-026 </td><td></td></tr><tr><td> Levamisol clorhidrato </td><td> Reactivo </td><td> ICN Biomedicals. Inc </td><td> 155228 </td><td></td></tr><tr><td> El ácido ribonucleico de la levadura torula, Tipo VI </td><td> Reactivo </td><td> Sigma </td><td> R6625 </td><td> fenol / cloroformo y precipitado en varias ocasiones, se resuspendió en DEPC dH2O y se almacenan a -20 ° C </td></tr><tr><td> Anti-digoxigenina-AP Fab fragmentos </td><td> Reactivo </td><td> Roche </td><td> 11093274910 </td><td></td></tr><tr><td> BM púrpura AP sustrato, lo que precipitó. Listo para usar la solución. </td><td> Reactivo </td><td> Roche </td><td> 11 442 074 001 </td><td></td></tr><tr><td> 4 ml, transparente, cerrado arriba, Vial de almacenamiento de conveniencia para la antorcha </td><td> Suministro </td><td> Científica nacional </td><td> B7800-2 </td><td></td></tr><tr><td> Shake N Hornee horno de hibridación </td><td> Equipo </td><td> Boekel Científico </td><td> 136400 </td><td></td></tr><tr><td> Biopsia del Seguro-Tek </td><td> Suministro </td><td> Fisherbrand </td><td> 15 a 200-402C </td><td></td></tr><tr><td> Además Paraplast tejido medio de inclusión </td><td> Reactivo </td><td> Fisherbrand </td><td> 23-021-400 </td><td></td></tr><tr><td> Pele-A-Way moldes de plástico desechables tejido incrustación </td><td> Suministro </td><td> Polysciences </td><td> 18646A </td><td></td></tr><tr><td> Además Colorfrost Portaobjetos </td><td> Suministro </td><td> Fisherbrand </td><td> 12-550-17 </td><td></td></tr><tr><td> Fast Red nuclear </td><td> Reactivo </td><td> Sigma </td><td> N8002 </td><td></td></tr><tr><td> Cytoseal 60 </td><td> Reactivo </td><td> Richard Allan-Científico </td><td> 8310-16 </td><td></td></tr></tbody></table>

whole mount in situ hybridization of e8.5 to e11.5 mouse embryos

Todo el montaje hibridación in situ es un enfoque muy informativo para la definición de patrones de expresión genética en los embriones. La hibridación in situ en los procedimientos son largos y técnicamente exigente con varios pasos importantes que en conjunto contribuyen a la calidad del resultado final. Este protocolo describe en detalle varios pasos clave de control de calidad para el etiquetado de la optimización de la sonda y el rendimiento. En general, el protocolo ofrece una descripción detallada de las medidas necesarias fundamental para obtener resultados de alta calidad reproducible. En primer lugar, se describe la generación de digoxigenina (DIG) marcado con sondas de ARN mediante transcripción in vitro de las plantillas de ADN generados por PCR. Se describen tres ensayos críticos de control de calidad para determinar la cantidad, la integridad y la actividad específica de la etiqueta DIG-sondas. Estos pasos son importantes para la generación de una sonda de la sensibilidad suficiente para detectar ARNm endógeno en un embrión de ratón entero. Además, se describen los métodos para la fijación y almacenamiento de E8.5-E11.5 embriones de ratón días de edad para la hibridación in situ. A continuación, se describen los métodos detallados para la digestión de proteinasa K limitado de los embriones rehidratado seguido de los detalles de las condiciones de hibridación, lavados post-hibridación y el tratamiento de RNasa para eliminar inespecíficos hibridación de la sonda. Un anticuerpo AP-conjugado se utiliza para visualizar la sonda marcada y revelar el patrón de expresión de la transcripción endógena. Los resultados representativos se muestran a partir de experiencias exitosas y las típicas experiencias óptimas.

Watch this Scientific Journal Video about Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos at JoVE.com

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Este monte se<em&gt; In situ</em&gt; Protocolo de hibridación se analizan los pasos críticos que garantizan resultados reproducibles de alta calidad para estudios de expresión génica en E8.5-E11.5 embriones de ratón días de edad.

Todo el monte In Situ La hibridación de E8.5 a E11.5 embriones de ratón

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

whole-mount-in-situ-hybridization-of-e85-to-e115-mouse-embryos

Research

JoVE Journal

Biology

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

25.8K vistas.  University of Georgia. Este monte se In situ Protocolo de hibridación se analizan los pasos críticos que garantizan resultados reproducibles de alta calidad para estudios de expresión génica en E8.5-E11.5 embriones de ratón días de edad.

Video: Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Todo el montaje utilizando In situ La hibridación de Enlace de Organismos y Biología Molecular

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Un alto rendimiento In situ Método de hibridación para caracterizar los patrones de expresión de ARNm en el ratón Fetal del tracto urogenital inferior

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Todo el monte ARN fluorescente<em&gt; In situ</em&gt; La hibridación de<em&gt; Drosophila</em&gt; Los embriones

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHibridación&gt; Los embriones en todo el montaje y la histología de células Adaptado para elasmobranquios marinos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolución todo el montaje de<em&gt; In Situ</em&gt; La hibridación en embriones de pez cebra para estudiar la expresión génica y la función

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje<em&gt; In situ</em&gt; La hibridación

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...