Los autores desean agradecer a Marika Walters para el asesoramiento en el desarrollo de la hibridación in situ en los métodos de los tejidos blandos y Goltsev Yury para la discusión de los protocolos para la hibridación in situ de Anopheles gambiae embriones, que fueron adaptados y modificados posteriormente para desarrollar el protocolo descrito aquí para la hibridación en in situ de Aedes y Culex embriones. Reconocemos también recomendaciones útiles dados por Adam Paré y Kosman David. Damos las gracias a Osvaldo Marinotti para la discusión científica y la edición del texto del protocolo.

<ol>
	<li>Juhn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+mapping+of+gene+expression+in+the+salivary+glands+of+the+dengue+vector+mosquito,+Aedes+aegypti.">Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, Aedes aegypti.</a> Parasit. Vectors. 4, 1 (2011).</li><li>Coleman, J., Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+midgut+and+salivary+glands+from+Ae.+aegypti+mosquitoes.">Dissection of midgut and salivary glands from Ae. aegypti mosquitoes.</a> J. Vis. Exp. (5), e228 (2007).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Plasmodium-infected+Mosquitoes:+Salivary+Glands,+Chapter.+5.4.2.">Dissecting Plasmodium-infected Mosquitoes: Salivary Glands, Chapter. 5.4.2.</a> Methods in Anopheles Research. , (2007).</li><li>Sappington, T. W., Brown, M. R., Raikhel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+analysis+of+insect+ovaries,+Chapter.+42.3.">Culture and analysis of insect ovaries, Chapter. 42.3.</a> The Molecular Biology of Insect Disease Vectors: A methods manual. , (1997).</li><li>Goltsev, Y., Hsiong, W., Lanzaro, G., Levine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+combinations+of+gap+repressors+for+common+stripes+in+Anopheles+and+Drosophila+embryos.">Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos.</a> Dev. Biol. 275, 435-446 (2004).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+Embryo+Fixation,+Chapter.+3.7.">Anopheles Embryo Fixation, Chapter. 3.7.</a> Methods in Anopheles Research. Malaria Research and Reference Reagent Resource Center (MR4). , (2007).</li><li>Juhn, J., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=oskar+gene+expression+in+the+vector+mosquitoes,+Anopheles+gambiae+and+Aedes+aegypti.">oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti.</a> Insect Mol. Biol. 15, 363-372 (2006).</li><li>Juhn, J., Marinotti, O., Calvo, E., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+structure+and+expression+of+nanos+(nos)+and+oskar+(osk)+orthologues+of+the+vector+mosquito,+Culex+quinquefasciatus.">Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus.</a> Insect Mol. Biol. 17, 545-552 (2008).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae).">Early Embryology of Aedes aegypti (L.) (Diptera: Culicidae).</a> Int. J. Insect Morphol. &#38; Embryol. 4, 517-528 (1975).</li><li>Raminani, L. N., Cupp, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryology+of+Aedes+aegypti+(L.)+(Diptera:+Culicidae):+Organogenesis.">Embryology of Aedes aegypti (L.) (Diptera: Culicidae): Organogenesis.</a> Int. J. Insect morphol. &#38; Embryol. 7, 273-296 (1978).</li><li>Ivanova-Kazas, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+Anopheles+maculipennis+Mg.">Embryonic development of Anopheles maculipennis Mg.</a> Izv. Akad. Nauk SSSR ser. Biol. 2, 140-170 (1949).</li><li>Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+evolutionary+basis+for+drought+tolerance+of+the+Anopheles+gambiae+embryo.">Developmental and evolutionary basis for drought tolerance of the Anopheles gambiae embryo.</a> Dev. Biol. 330, 462-470 (2009).</li><li>Papatsenko, D., Levine, M., Goltsev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clusters+of+temporal+discordances+reveal+distinct+embryonic+patterning+mechanisms+in+Drosophila+and+Anopheles.">Clusters of temporal discordances reveal distinct embryonic patterning mechanisms in Drosophila and Anopheles.</a> PLoS Biol. 9, e1000584 (2011).</li><li>Davis, C. 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=A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+(Diptera:+Culicidae)+and+the+sheep-fly+Lucilla+seriata+Meigen+(Diptera:+Calliphoridae)+I.+Description+of+embryonic+development.">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilla seriata Meigen (Diptera: Calliphoridae) I. Description of embryonic development.</a> Aust. J. Zool. 15, 547-579 (1967).</li></ol>

Los autores no tienen revelaciones.

La hibridación in situ y el protocolo de tinción colorimétrica que aquí se presenta para los tejidos de mosquitos todo el montaje y los embriones es una técnica útil para la localización de las transcripciones en órganos específicos y tipos de células. Estos procedimientos son una mejora con respecto a los métodos ya se ha informado, tanto en la racionalización de amplias medidas de lavado y de proporcionar más detalles técnicos y las fuentes de reactivos. En nuestra experiencia, la detección colorimétrica de las señales de hibridación es superior en sensibilidad y la claridad de la señal de hibridación en comparación con esquemas de detección basadas en fluorescencia. Por otra parte la detección colorimétrica elude las cuestiones relacionadas con la discriminación de la señal en los embriones, que son inherentemente auto-fluorescente. Las limitaciones en la detección de señales de hibridación ocurre, cuando baja abundancia transcripciones están dirigidos y tinción de fondo es evidente. El aumento de la temperatura de hibridación a 65 ° C se ha encontrado para reducir de nuevoseñales de hibridación de tierra, pero no es un sustituto sugerido para el diseño único objetivo sondas específicas de ARN. Este protocolo se ha utilizado para llevar a cabo la hibridación in situ de todo el montaje las glándulas salivales del mosquito Aedes aegypti y los ovarios y los embriones de Anopheles gambiae, el Anopheles stephensi, Ae. aegypti y Culex quinquefasciatus. Este método también es aplicable a los tejidos de mosquitos otros, y presumiblemente los de otros insectos. Además, se compiló para las pautas de la primera vez comparativas para la puesta en escena del desarrollo embrionario de cada tres mosquitos vectores, Ae. aegypti, Un. gambiae y Cx. fatigans. Las observaciones se han reportado para cepas específicas de estas tres especies, bajo condiciones de cría discretos. Es importante señalar que el curso de tiempo de desarrollo puede variar para diferentes cepas de especies de mosquitos y bajo condiciones de cría diferentes. Hibridaciones en el suplemento in situ los esfuerzos en curso a analisar los transcriptomes de mosquitos y otros artrópodos, y puede ofrecer una mejor imagen de la regulación de la expresión génica en estos organismos.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> EDTA 0,5 M </td><td> Ambion </td><td> AM9261 </td><td></td></tr><tr><td> Tris-HCl 1M </td><td> Ambion </td><td> AM9855G </td><td> pH 8,0 </td></tr><tr><td> PBS 10 veces </td><td> Ambion </td><td> AM9625 </td><td></td></tr><tr><td> SSC 20X </td><td> Ambion </td><td> AM9763 </td><td></td></tr><tr><td> 1,5 ml tubos de microfuga </td><td> Ambion </td><td> AM12400 </td><td> Menos opaco que los tubos estándar, ayuda a visualizar las muestras </td></tr><tr><td> Formamida desionizada </td><td> Ambion </td><td> AM9342 </td><td> El almacenamiento a 4 ° C </td></tr><tr><td> DEPC agua </td><td> Ambion </td><td> AM9932 </td><td></td></tr><tr><td> Proteinasa K </td><td> Ambion </td><td> AM2546 </td><td></td></tr><tr><td> 5,25% de hipoclorito de sodio </td><td> Austin </td><td></td><td> A-1 Marca </td></tr><tr><td> T3 ARN polimerasa-Plus </td><td> Ambion </td><td> AM2733 </td><td> El almacenamiento a -20 ° C </td></tr><tr><td> T7 ARN polimerasa </td><td> Ambion </td><td> AM2082 </td><td> El almacenamiento a -20 ° C </td></tr><tr><td> 95% de etanol </td><td> Fisher Scientific </td><td> AC61511-0040 </td><td></td></tr><tr><td> Fisherbrand pipetas desechables de polietileno de transferencia </td><td> Fisher Scientific </td><td> 13-711-7M </td><td></td></tr><tr><td> 37% de formaldehído </td><td> Fisher Scientific </td><td> F79-500 </td><td></td></tr><tr><td> HPLC-grado de metanol </td><td> Fisher Scientific </td><td> A452-1 </td><td></td></tr><tr><td> Cloruro de magnesio </td><td> Fisher Scientific </td><td> M87-500 </td><td></td></tr><tr><td> Microscopio cubierta de vidrio </td><td> Fisher Scientific </td><td> 12-542A </td><td> 18 x 18 mm </td></tr><tr><td> N-heptano </td><td> Fisher Scientific </td><td> H350-1 </td><td></td></tr><tr><td> P-xileno </td><td> Fisher Scientific </td><td> O5082-500 </td><td></td></tr><tr><td> Pyrex de 9 y Punto Plate </td><td> Fisher Scientific </td><td> 13-748B </td><td> 100x85 mm </td></tr><tr><td> Cloruro de sodio </td><td> Fisher Scientific </td><td> AC32730-0025 </td><td></td></tr><tr><td> Hidróxido de sodio </td><td> Fisher Scientific </td><td> SS255-1 </td><td></td></tr><tr><td> Superfrost / Plus portaobjetos </td><td> Fisher Scientific </td><td> 12-550-15 </td><td> 25x75x1.0 mm </td></tr><tr><td> Davlyn Red Clear-liner Toupee cinta </td><td> Cabello directo </td><td> RED-75R12 </td><td> Poli / material de la piel base de 0,75 cm x 12 m rollo de cinta </td></tr><tr><td> Kit de Clonación de TOPOTA Sequening con un disparo Top10 químicamente competente E. E. </td><td> Invitrogen </td><td> K457501 K457540 </td><td> 20 reacciones 40 reacciones </td></tr><tr><td> De salmón sonicado ADN de los espermatozoides </td><td> Invitrogen </td><td> 15632-011 </td><td> El almacenamiento a -20 ° C </td></tr><tr><td> Anti-digoxigenina-AP Fab </td><td> Roche Ciencias Aplicadas </td><td> 1093274 </td><td> El almacenamiento a 4 ° C </td></tr><tr><td> DIG RNA etiquetado mezcla </td><td> Roche Ciencias Aplicadas </td><td> 1277073 </td><td> El almacenamiento a -20 ° C </td></tr><tr><td> NBT / BCIP de la solución madre </td><td> Roche Ciencias Aplicadas </td><td> 1681451 </td><td> El almacenamiento a 4 ° C </td></tr><tr><td> Reactivo de bloqueo occidental </td><td> Roche Ciencias Aplicadas </td><td> 11921673001 </td><td> El almacenamiento a 4 ° C </td></tr><tr><td> Saatilene Hitech malla de poliéster (330.130) </td><td> Saati Imprimir </td><td> 330,34 UO PW </td><td> 330 hilos por pulgada, de 34 micras de diámetro de la rosca, color naranja </td></tr><tr><td> Glicerol </td><td> Sigma </td><td> G6279-1 </td><td> 70% en PBT </td></tr><tr><td> Sal de sodio de heparina </td><td> Sigma </td><td> H3393 </td><td></td></tr><tr><td> Tween 20 </td><td> Sigma </td><td> P1379-500 </td><td></td></tr><tr><td> 37% de formaldehído </td><td> Ted Pella </td><td> 18508 </td><td> Alícuotas de 10 ml en ampollas ámbar </td></tr><tr><td> 16 oz Recipientes con tapas de papel solitario </td><td> El Paper Company </td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td> Vidrio borosilicato scintillatión vial con tapón de rosca sin ataduras </td><td> VWR International </td><td> 66022-128 </td><td> 20 ml caso de 500 </td></tr><tr><td> Sealed Air Bubble Wrap o via un material de relleno </td><td> VWR International </td><td> 500018-081 </td><td> De 10 pies / rollo 0.188 pulgadas de espesor </td></tr><tr><td> Suero de pollo </td><td></td><td></td><td> Toda la sangre se recoge ya sea desde la vena del ala, o por punción cardiaca de un pollo de menores. La sangre se incubó a 37 ° C durante 1 h hasta coagulada y luego se coloca en hielo durante 30 min. El suero se recogió y se centrifugó a 3000 xg durante 10 min. El sobrenadante resultante (aclarado suero) se recogió y se almacenó a -20 ° C hasta su uso. </td></tr></tbody></table> Tabla 4. Tabla de los reactivos específicos y equipos para la hibridación in situ.

hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes

Los mosquitos son vectores de un conjunto diverso de agentes patógenos como los arbovirus, parásitos protozoarios y nematodos. Investigación de las transcripciones y los reguladores de genes que se expresan en tejidos en los que el anfitrión de mosquitos e interactuar patógeno, y en los órganos implicados en la reproducción son de gran interés para las estrategias para reducir la transmitida por el mosquito transmisión de la enfermedad e interrumpir el desarrollo del óvulo. Un número de herramientas se han empleado para estudiar y validar la regulación temporal y tejidos específicos de la expresión génica. A continuación, describimos los protocolos que han sido desarrollados para obtener la información espacial, lo que aumenta nuestra comprensión de los genes específicos en donde se expresan y acumulan sus productos. El protocolo descrito se ha utilizado para validar la expresión y de determinar los patrones de acumulación de expedientes en los tejidos relacionados con la transmitida por el mosquito transmisión de patógenos, tales como las mujeres las glándulas salivales, así como los compartimentos subcelulares de los ovarios y los embriones, que vuelva atarde para la reproducción de mosquitos y el desarrollo. Los procedimientos siguientes representan una metodología optimizada que mejora la eficiencia de varios pasos en el protocolo sin pérdida de las señales de hibridación específicos diana. Directrices para la preparación de la sonda de ARN, la disección de los tejidos blandos y el procedimiento general para la fijación y la hibridación se describen en la Parte A, mientras que los pasos específicos para la recogida, grabación, pre-hibridación y la hibridación de embriones de mosquitos se detallan en la Parte B.

Watch this Scientific Journal Video about Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes at JoVE.com

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Análisis de genes temporales y espaciales de expresión tiene un papel crucial en la genómica funcional. Todo el montaje hibridación<em&gt; In situ</em&gt; Es útil para determinar la localización de las transcripciones dentro de los tejidos y los compartimentos subcelulares. Aquí describimos una hibridación<em&gt; In situ</em&gt; Protocolo con las modificaciones de los tejidos diana específicos en los mosquitos.

Hibridación<em&gt; In situ</em&gt; De las glándulas salivales, los ovarios y embriones de los mosquitos vectores

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

hybridization-situ-salivary-glands-ovaries-embryos-vector

Research

JoVE Journal

Immunology and Infection

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

16.4K vistas.  University of California, Irvine. Análisis de genes temporales y espaciales de expresión tiene un papel crucial en la genómica funcional. Todo el montaje hibridación In situ Es útil para determinar la localización de las transcripciones dentro de los tejidos y los compartimentos subcelulares. Aquí describimos una hibridación In situ Protocolo con las modificaciones de los tejidos diana específicos en los mosquitos.

Video: Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detección del virus infeccioso de campo recogidos por los mosquitos del ensayo cultivo celular Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescente<em&gt; In situ</em&gt; La hibridación en los cromosomas mitóticos de mosquitos

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Un método para la medición de la función de la glándula salival en ratones

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Fluorescencia cuantitativa en hibridación situ (FISH) e inmunofluorescencia (IF) de productos genéticos específicos en células infectadas por KSHV

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

Aislamiento de células epiteliales salivales de glándulas salivales humanas para el crecimiento in vitro como salisferas o monocapas

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualización de Candida albicans en el tracto gastrointestinal murino utilizando hibridación fluorescente in situ

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Análisis de competencia vectorial en mosquitos Aedes aegypti con el virus Zika

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

Visualización Del SARS-CoV-2 Usando Inmuno ARN-Fluorescencia Hibridación In Situ

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...