Os autores gostariam de agradecer a Marika Walters para o conselho de hibridização desenvolvimento de métodos in situ de tecidos moles e Goltsev Yury para discussão de protocolos para a hibridização in situ de embriões Anopheles gambiae, que foram posteriormente adaptados e modificados para desenvolver o protocolo descrito aqui para hibridização in situ de Aedes e Culex embriões. Reconhecemos também recomendações úteis dadas por Adam Paré e Kosman David. Agradecemos Osvaldo Marinotti para a discussão científica e editar o texto do 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>

Os autores não têm divulgações.

A hibridização in situ protocolo de coloração e colorimétricas apresentadas aqui para tecidos toda montagem de mosquitos e de embriões é uma técnica útil para a localização de transcritos dentro dos órgãos e tipos específicos de células. Esses procedimentos são um avanço em relação nossos métodos reportados anteriormente, tanto na racionalização etapas de lavagem e proporcionando amplos detalhes técnicos adicionais e fontes de reagentes. Na nossa experiência, a detecção colorimétrica de sinais de hibridação é superior em sensibilidade e clareza de sinal de hibridação em comparação com os regimes de fluorescência de detecção baseados em. Além disso detecção colorimétrica contorna problemas associados com a discriminação do sinal em embriões, que são inerentemente auto-fluorescente. Limitações na detecção de sinais de hibridação ocorrer, quando baixa abundância transcrições são alvejados e coloração de fundo é evidente. O aumento da temperatura de hibridação a 65 ° C, foi encontrada a reduzir para trássolo sinais de hibridação, mas não é um substituto sugerido para a concepção de sondas único alvo-específicas de RNA. Este protocolo tem sido usado para realizar a hibridação in situ de toda montagem glândulas salivares de Aedes aegypti, e os ovários e embriões de Anopheles gambiae, Anopheles stephensi, Ae. aegypti e Culex quinquefasciatus. Este método também é aplicável a outros tecidos de mosquito, e presumivelmente aqueles de outros insectos. Além disso, nós compilamos para as diretrizes da primeira vez comparativas para a realização do desenvolvimento embrionário em três mosquitos vetores, Ae. aegypti, An. gambiae e Cx. fatigans. Observações foram relatadas para as estirpes específicas destas três espécies, em condições de criação discretos. É importante notar que o curso de tempo de desenvolvimento podem variar para diferentes estirpes de espécies de mosquitos e sob condições de criação diferentes. Hibridizações em suplemento situ em curso esforços para analisar as transcriptomas de mosquitos e outros artrópodes, e pode fornecer uma melhor imagem da regulação da expressão genética nestes organismos.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> EDTA 0,5 M </td><td> Ambion </td><td> AM9261 </td><td></td></tr><tr><td> 1M Tris-HCl </td><td> Ambion </td><td> AM9855G </td><td> pH 8,0 </td></tr><tr><td> PBS 10X </td><td> Ambion </td><td> AM9625 </td><td></td></tr><tr><td> 20X SSC </td><td> Ambion </td><td> AM9763 </td><td></td></tr><tr><td> Microcentrífuga de 1,5 ml tubos </td><td> Ambion </td><td> AM12400 </td><td> Menos opaco do que os tubos padrão; auxilia na visualização de amostras </td></tr><tr><td> Formamida deionizada </td><td> Ambion </td><td> AM9342 </td><td> O armazenamento a 4 ° C </td></tr><tr><td> Água DEPC </td><td> Ambion </td><td> AM9932 </td><td></td></tr><tr><td> Proteinase K </td><td> Ambion </td><td> AM2546 </td><td></td></tr><tr><td> O hipoclorito de sódio 5,25% </td><td> Austin </td><td></td><td> A-1 Marca </td></tr><tr><td> RNA polimerase T3 Plus- </td><td> Ambion </td><td> AM2733 </td><td> O armazenamento a -20 ° C </td></tr><tr><td> T7 RNA Polimerase </td><td> Ambion </td><td> AM2082 </td><td> O armazenamento a -20 ° C </td></tr><tr><td> Etanol a 95% </td><td> Fisher Scientific </td><td> AC61511-0040 </td><td></td></tr><tr><td> Fisherbrand pipetas de transferência de polietileno descartáveis </td><td> Fisher Scientific </td><td> 13-711-7M </td><td></td></tr><tr><td> Formaldeído a 37% </td><td> Fisher Scientific </td><td> F79-500 </td><td></td></tr><tr><td> HPLC metanol </td><td> Fisher Scientific </td><td> A452-1 </td><td></td></tr><tr><td> Cloreto de magnésio </td><td> Fisher Scientific </td><td> M87-500 </td><td></td></tr><tr><td> Tampa de vidro de microscópio </td><td> Fisher Scientific </td><td> 12-542A </td><td> 18 x18 milímetros </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 Placa Ponto 9-bem </td><td> Fisher Scientific </td><td> 13-748B </td><td> 100x85 milímetros </td></tr><tr><td> Cloreto de sódio </td><td> Fisher Scientific </td><td> AC32730-0025 </td><td></td></tr><tr><td> De hidróxido de sódio </td><td> Fisher Scientific </td><td> SS255-1 </td><td></td></tr><tr><td> SuperFrost / Plus lâminas de microscópio </td><td> Fisher Scientific </td><td> 12-550-15 </td><td> 25x75x1.0 milímetros </td></tr><tr><td> Davlyn Red fita Toupee-liner Limpar </td><td> Cabelo Direto </td><td> RED-75R12 </td><td> Poly / material Pele base de 0,75 em x rolo de fita 12 m </td></tr><tr><td> TOPOTA Kit clonagem para Sequening com um tiro Top10 quimicamente competente E. coli </td><td> Invitrogen </td><td> K457501 K457540 </td><td> 20 reacções 40 reações </td></tr><tr><td> ADN de esperma de salmão sonicado </td><td> Invitrogen </td><td> 15632-011 </td><td> O armazenamento a -20 ° C </td></tr><tr><td> Anti-digoxigenina-AP fragmentos Fab </td><td> Roche Applied Science </td><td> 1093274 </td><td> O armazenamento a 4 ° C </td></tr><tr><td> DIG RNA rotulagem mix </td><td> Roche Applied Science </td><td> 1277073 </td><td> O armazenamento a -20 ° C </td></tr><tr><td> NBT / BCIP de solução stock </td><td> Roche Applied Science </td><td> 1681451 </td><td> O armazenamento a 4 ° C </td></tr><tr><td> Reagente de bloqueio Ocidental </td><td> Roche Applied Science </td><td> 11921673001 </td><td> O armazenamento a 4 ° C </td></tr><tr><td> Saatilene Hitech malha de poliéster (330,130) </td><td> Saati impressão </td><td> 330,34 UO PW </td><td> 330 fios / cm, 34 micron diâmetro de rosca, cor de laranja </td></tr><tr><td> Glicerina </td><td> Sigma </td><td> G6279-1 </td><td> 70% em PBT </td></tr><tr><td> Sal de sódio da 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> Formaldeído a 37% </td><td> Ted Pella </td><td> 18508 </td><td> Alíquotas de 10 ml em ampolas âmbar </td></tr><tr><td> 16 onças Individuais recipientes de papel com tampas </td><td> O Paper Company </td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td> Vidro borosilicato scintillatfrasco de íons com tampa de rosca solta </td><td> VWR International </td><td> 66022-128 </td><td> 20 ml de 500 caso </td></tr><tr><td> Sealed Air Bubble Wrap material de enchimento celluar </td><td> VWR International </td><td> 500018-081 </td><td> 10 pés / roll 0,188 polegadas de espessura </td></tr><tr><td> Frango soro </td><td></td><td></td><td> O sangue total foi recolhido quer a partir da veia da asa ou por punção cardíaca a partir de uma galinha juvenil. O sangue foi incubada a 37 ° C durante 1 h até coagulado e depois colocado em gelo durante 30 min. O soro foi recolhido e centrifugado a 3000 xg durante 10 min. O sobrenadante resultante (clarificado soro) foi recolhido e armazenado a -20 ° C até à utilização. </td></tr></tbody></table> Tabela 4. Tabela de reagentes específicos e equipamento para a hibridação in situ.

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

Os mosquitos são vetores para um conjunto diverso de patógenos, incluindo arbovírus, protozoários e nematóides. Investigação de transcrições e reguladores de genes que são expressos em tecidos em que o mosquito hospedeiro e interagir patógeno, e de órgãos envolvidos na reprodução são de grande interesse para as estratégias para reduzir o mosquito-borne transmissão da doença e impedir o desenvolvimento do ovo. Um certo número de ferramentas foram utilizados para estudar e validar a regulação temporal e tecido específico da expressão do gene. Aqui, nós descrevemos protocolos que têm sido desenvolvidos para obter informação espacial, que aumenta a nossa compreensão de onde genes específicos são expressos e os seus produtos acumular. O protocolo descrito foi utilizado para validar expressão e determinar os padrões de acumulação de transcritos em tecidos relacionados com transmitida por mosquitos transmissão de patógenos, tais como do sexo feminino glândulas salivares, bem como compartimentos subcelulares de ovários e embriões, que retarde para reprodução do mosquito e desenvolvimento. Os procedimentos seguintes representam uma metodologia optimizado que melhora a eficiência de vários passos do protocolo sem perda de sinais de alvo específicas de hibridação. Diretrizes para a preparação do RNA sonda, a dissecção dos tecidos moles e ao procedimento geral para a fixação e hibridação estão descritos na Parte A, enquanto os passos específicos para a coleta, fixação, hibridação e pré-hibridação de embriões de mosquitos são detalhados na 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

Análises de expressão temporal e espacial de genes têm um papel crucial em genômica funcional. Todo mount-hibridização<em&gt; In situ</em&gt; É útil para determinar a localização de transcritos nos tecidos e compartimentos subcelulares. Aqui nós esboçamos uma hibridização<em&gt; In situ</em&gt; Protocolo com modificações para tecidos-alvo específicos em mosquitos.

Hibridização<em&gt; In situ</em&gt; Das glândulas salivares, ovários e embriões de mosquitos vetores

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

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JoVE Journal

Immunology and Infection

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

16.5K visualizações.  University of California, Irvine. Análises de expressão temporal e espacial de genes têm um papel crucial em genômica funcional. Todo mount-hibridização In situ É útil para determinar a localização de transcritos nos tecidos e compartimentos subcelulares. Aqui nós esboçamos uma hibridização In situ Protocolo com modificações para tecidos-alvo específicos em mosquitos.

Vídeo: 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).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de 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 ...

Imunologia e Infecção

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.

Detecção de vírus infecciosos de Campo mosquitos coletados por Vero Ensaio Cultura de Células

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.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos 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; Hibridação em cromossomos 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. ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Pintura de cromossomo 2D e 3D em mosquitos da malária

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

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.

Um método para a medição da função da glândula salivar em ratos

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).

Hibridação in situ da fluorescência quantitativa (peixes) e immunofluorescence (se) de produtos específicos do gene em pilhas KSHV-infectadas

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.

Isolação de pilhas epithelial salivares das glândulas salivares humanas para o crescimento in vitro como Salispheres ou 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 ...

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.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

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.

Análises de competência vetorial sobre mosquitos Aedes aegypti que usam zika vírus

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

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