Questo lavoro è stato supportato dai numeri concedere R01AI055001 e R01AI082127 dal NIAID di NRM e Premio tesi SREB Fellowship di JLB.

1. Fluorescina etichettata destrano e GSL I-isolectin B 4 iniezioni vena facciale per etichettare vasi embrionali <ol><li> Preparare FITC-destrano (50ug/mL) in soluzione tampone fosfato (PBS) o GSL 1 - isolectin B 4 (20ug/200uL) in PBS in un tubo Eppendorf 1,5 ml e caldo a 37 ° C. Aggiungi 100uL di magazzino 1,25 mm Fast Green / PBS al FITC-destrano soluzione (volume totale 1 ml) e 180uL di azioni da 1,25 mm Fast Green / PBS per la GSL 1 - isolectin B 4 (200uL volume totale), in modo che la soluzione è visibilmente blu. </li><li> Sezionare E14.5 E18.5-embrioni e sacco vitellino insieme, lasciando il gambo allantoide (arteria e vena ombelicale) intatti. </li><li> Trasferimento degli embrioni per un nuovo piatto Petrie (60 X 15 mm) e immergerli in PBS a temperatura ambiente. </li><li> Posizionare l&#39;embrione per fornire una visione sagittale della testa / viso. Usa micro dissettore ad afferrare delicatamente l&#39;embrione alla testa. </li><li> Utilizzando un ago 30G, iniettare 50uL FITC-destrano (50ug/mL) o GSL 1 - isolectin B 4 (in 20ug 200uL PBS) nella vena del viso che punta l&#39;ago verso la parte posteriore della testa. </li><li> Quando il colorante è visibile nella vena ombelicale, rimuovere l&#39;ago e separare l&#39;embrione dal gambo allantoide (arteria e vena ombelicale). </li><li> A seguito di iniezioni, permette all&#39;embrione di rimanere in PBS a temperatura ambiente per 2-3 minuti in modo che il colorante circola in tutto l&#39;embrione. </li></ol> 2. Tutto-mount analisi di vascolarizzazione della pelle <ol><li> Dopo aver lasciato la tintura di circolare in tutta l&#39;embrione, rimuovere campioni di pelle da regioni degli arti, alla schiena e allo stomaco, ecc 8,9. </li><li> Lavare campione di pelle in PBS freddo, e fissare nel 4% PFA / PBS per 2 ore 8,9. Lavare 3 volte per 10 minuti ogni 4 ml in flaconcino trasparente con 2 ml PBS freddo. </li><li> Pelle del campione posto su un vetrino da microscopio e aggiungere 100 ml di montaggio di mezzi di ogni diapositiva e un vetro di copertura. </li><li> Lasciare i vetrini si asciughino in una zona di conservazione al buio. </li><li> Procedere al passaggio sezione &#39;acquisizione di immagini&#39; 2. </li></ol> 3. Multi-color etichettatura di timo e vascolarizzazione del cuore e le cellule perivascolari per criosezioni (Continua dalla sezione 1, punto 7) <ol><li> &#39;Flash congelare&#39; embrione intero in azoto liquido. Gli embrioni possono essere e conservati a -80 ° C fino al momento dell&#39;analisi. </li><li> In alternativa, sezionare fuori timo, sciacquare in 4 ° C PBS, e fissare in 2 ml paraformaldeide 4% (PFA) / PBS per 2 ore. Lavare 3 volte per 10 minuti in PBS freddo, posto Thymi in ottobre, e congelare e conservare fino al momento dell&#39;uso a -80 ° C. </li><li> Per criosezionamento, diffusione ottobre su &#39;block&#39; una sezione e montare l&#39;embrione o di organi sezionati / tessuti per sezionamento. </li><li> Tagliare tessuti congelati in 10 sezioni micron di spessore e raccogliere su vetrini. </li><li> Fix sezioni in acetone per 5-10 minuti. Lavare 3 volte in TBS freddo. </li><li> Blocco in asino siero del 10% / TBS in una camera umida a temperatura ambiente. </li><li> Incubare sezioni per 1 ora, durante la notte con 100 ml di anticorpi primari in una camera umida a 4 ° C: in questo esempio, utilizziamo ratto anti-CD31 topo (1:100) all&#39;endotelio etichetta, e di capra anti-topo PDGFR-β (1:100) per etichettare le cellule perivascolari. E &#39;utile per coprire le diapositive con le strisce Parafilm tagliati singolarmente per garantire che l&#39;anticorpo è uniformemente diffuso in tutta la sezione. </li><li> In seguito a incubazione con anticorpo primario, lavare le sezioni 3 volte in TBS freddo. Incubare con 100 ml di appropriati anticorpi secondari per 30 minuti minimo. </li><li> Lavare 3 volte in TBS freddo. Aggiungere 100 ml di montaggio di mezzi di ogni diapositiva e un vetro di copertura. </li><li> Lasciare i vetrini si asciughino in una zona di conservazione al buio. </li><li> Passare alla sezione &#39;acquisizione di immagini&#39;. </li></ol> 4. Multi-color etichettatura di timo vasi e cellule perivascolari per le sezioni vibratome (Continua dalla sezione 1, punto 7) <ol><li> Sezionare i lobi del timo dall&#39;embrione e lavare in PBS freddo. </li><li> Fissare timo nel 4% PFA / PBS a temperatura ambiente per 2 ore. </li><li> Lavare in PBS-Triton X (0,15%) 3 volte, 10 minuti e Thymi posto in una cartuccia di plastica e immergere nel 4% bassa temperatura di fusione agarosio / PBS (~ 4 ° C). Il timo deve essere in contatto con il fondo della cartuccia. </li><li> Lasciare solidificare agarosio su ghiaccio (3-5 minuti). Utilizzare una lama di rasoio per tagliare l&#39;eccesso di agarosio. Aggiungere la colla al blocco vibratome e aderire al blocco del campione. </li><li> Aggiungi PBS freddo vibratome a bagnomaria fino a quando il campione e la lama sono immersi. </li><li> Impostare la velocità e l&#39;ampiezza (ampiezza alta e bassa-moderata velocità è ideale per le sezioni morbide timo). L&#39;ampiezza dovrebbe essere ridotto se le sezioni rompere a causa di agitazione in eccesso. </li><li> Taglio del 50 sezioni um. </li><li> Utilizzando un pennello, raccogliere le sezioni di un 24-bene micropiastre in PBS freddo. </li><li> Sezioni di blocco in 500 ml di siero di asino al 10% in PBS-Triton X (0,15%) per 30 minuti. </li><li> Sezioni incubare per 8 ore a notte con l&#39;anticorpo primario, come anti-CD31 e anti-PDGFR-β, In una coperta di 24 e micropiastre. </li><li> Lavare 3 volte in PBS-Triton X (0,15%) su un totale di 8 ore a 4 ° C. </li><li> Sezioni di blocco nel siero asino 10% in PBS-Triton X (0,15%) per 30 minuti. </li><li> Incubare sezioni per 8 ore per notte a 4 ° C con anticorpi secondaria appropriata. </li><li> Lavare 3 volte in PBS-Triton X (0,15%) su un totale di 8 ore a 4 ° C. </li><li> Ri-fix campioni nel 4% PFA / PBS per 30 minuti in ghiaccio. </li><li> Lavare 3 volte in PBS-Triton X (0,15%) più di 30 minuti sul ghiaccio. </li><li> Disidratare campioni attraverso un graduale MeOH / PBS-Triton X serie: 25% MeOH, 50% MeOH, 75% MeOH, e 100% MeOH a 10 minuti per ogni passaggio. Sostituire il 100% MeOH con MeOH fresca dopo 10 minuti e incubare per 1 ora a temperatura ambiente. </li><li> In un contenitore di vetro, mescola BABB (Benzyl Alcohol: Benzyl Benzoate) in un rapporto 1:2. Combina BABB con MeOH per una concentrazione finale del 50% e 50% BABB MeOH. Incubare il campione in BABB: MeOH per 10-15 minuti. </li><li> Trasferimento del campione a un contenitore di vetro con% BABB 100 e incubare per 10-15 minuti o finché non viene chiusa, a temperatura ambiente. </li><li> Compila scivolo depressione (0,7 millimetri di profondità) con fresca BABB 100% e di trasferimento del campione a lato. Aggiungi coperchio in vetro (n. 1.5) e sigillare con 2-3 mani di smalto. Lasciare smalto per indurire al buio a temperatura ambiente, quindi memorizzare campione a 4 ° C. Nota: Le diapositive devono essere completamente sigillati prima di acquisizione di immagini confocale. Le immagini devono essere acquisite entro 12-24 ore, come i coloranti fluorescenti possono sbiadire a Babb. </li><li> Passare alla sezione &#39;acquisizione di immagini&#39;. </li></ol> 5. Acquisizione di immagini <ol><li> Immagine 10 micron sezioni congelate con un microscopio confocale utilizzando il Plan-Apochromat 20X/0.8 obiettivo (512 x 512 pixel) con 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 - e 633-nm linee laser. </li><li> Acquisire confocale z-sezioni di pelle monte intero e 50 micron agarosio-embedded sezioni utilizzando il Plan-Apochromat 10X/0.4 obiettivo (512 x 512 pixel) con 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 -, e 633-nm linee laser. Serie Z-sezioni devono essere raccolti in sequenza al numero 1-micron per ogni rispettivo canale. </li><li> Ricostruire serie Z-sezioni utilizzando Zeiss AxioVision 4.6 o altri software di analisi dell&#39;immagine. </li></ol> 6. Rappresentante dei risultati: Efficace di etichettatura dei vasi embrionali è fondamentale per valutare i difetti dei vasi sanguigni nei topi embrionale. La figura 1 mostra l&#39;etichettatura specifica E16.5 vasi sanguigni del timo (1A-B) e co-marcatura con CD31 (1B), oltre alla colorazione dei ventricoli destro e sinistro (1E-F), rispettivamente. Il GSL I-isolectin B 4 protocollo per criosezioni come descritto nelle sezioni 1, 3 e 5 è stato usato in questi esperimenti. Tutto-mount etichettatura della rete dei vasi sanguigni della pelle su E16.5 topi, utilizzando i protocolli descritti nelle sezioni 1, 2 e 5 è mostrato nella figura 1C-D. <img alt="Figura 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> Figura 1 Legend. FITC GSL I - isolectin B 4 iniezioni in vena facciale E16.5 embrioni di topo. Cryosection a. timo embrionale dopo l&#39;iniezione. b. Fusione di CD31 co-etichettatura con isolectin B 4. c. e d. Whole-mount dell&#39;iniezione embrionale vascolarizzazione della pelle successivo. e. ed f. Cryosection embrionale di cuore e. (a destra ventricolo) f. (ventricolo sinistro) dopo l&#39;iniezione.

<ol>
	<li>Cuddihy, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-mediated+cross-talk+within+the+neonatal+murine+thymus.">VEGF-mediated cross-talk within the neonatal murine thymus.</a> Blood. 113, 2723-2731 (2009).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+of+VEGF-A+in+thymus+epithelium+disrupts+thymus+blood+vessel+architecture.">Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 10587-10592 (2005).</li><li>Muller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+origin+of+perivascular+mesenchyme+in+the+adult+thymus.">Neural crest origin of perivascular mesenchyme in the adult thymus.</a> J. Immunol. 180, 5344-5351 (2008).</li><li>Foster, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+neural+crest-derived+cells+in+the+embryonic+and+adult+thymus.">Contribution of neural crest-derived cells in the embryonic and adult thymus.</a> J. Immunol. 180, 3183-3189 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+between+CCR7-+and+CCR9-mediated+chemokine+signals+in+prevascular+fetal+thymus+colonization.">Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.</a> Blood. 108, 2531-2539 (2006).</li><li>Lavine, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Lavine, K. J., Kovacs, A., Ornitz, 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=Hedgehog+signaling+is+critical+for+maintenance+of+the+adult+coronary+vasculature+in+mice.">Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice.</a> J. Clin Invest. 118, 2404-2414 (2008).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Whole-mount e PECAM-1 (CD31) colorazione sulle sezioni sono i metodi convenzionali per l&#39;etichettatura del sistema vascolare nei topi embrionale. Questi metodi richiedono l&#39;uso di immunofluorescenza diretta e / o indiretta, e detergenti per permeabilize tessuto del mouse. Questo risulta essere un processo piuttosto tempestivo. Qui, abbiamo impiegato FITC-destrano o isolectin B 4 iniezioni vena facciale per etichettare direttamente la vascolarizzazione embrionali, eliminando così la necessità di fasi...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> FITC-destrano </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Fluorescina etichettata GSL 1 - isolectin B 4 </td><td> Laboratori Vector </td><td> FL-1201 </td></tr><tr><td> Fast Green </td><td> MP Biomedicals </td><td> 195178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76240 </td></tr><tr><td> Siero bovino fetale </td><td> Atlanta Biologicals </td><td> S11550 </td></tr><tr><td> Temperatura ottimale composto da taglio (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> Acetone </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Donkey Siero </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> topo anti-topo CD31, </td><td> BD Pharmingen </td><td> 558736 </td></tr><tr><td> capra anti-topo PDGFR-β </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> asino anti-ratto CD31 Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> asino anti-capra Alexa 594 (Invitrogen) </td><td> Invitrogen </td><td> A11058 </td></tr><tr><td> Triton X -100 </td><td> Sigma-Aldrich </td><td> X-100 </td></tr><tr><td> Bassa temperatura di fusione agarosio / PBS </td><td> Sigma-Aldrich </td><td> A9414-25G </td></tr><tr><td> Metanolo </td><td> Fisher Scientific </td><td> A413-4 </td></tr><tr><td> L&#39;alcol benzilico </td><td> Acros scientifico </td><td> 148390010 </td></tr><tr><td> Benzyl Benzoate </td><td> Acros scientifico </td><td> 105860010 </td></tr><tr><td> Depressione diapositive </td><td> Fisher Scientific </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> Electron Microscopy Sciences </td><td> 17985-10 </td></tr><tr><td> Copertura di vetro (22x22) -1,5 </td><td> Thermo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Meta microscopio confocale </td><td> Zeiss </td><td></td></tr><tr><td> Micro dissettore </td><td> Roboz </td><td> RS-5135 </td></tr><tr><td> Parafilm No. OM992 </td><td> Fisher Scientific </td><td> 13-374-16 </td></tr><tr><td> 12 e 24 micropiastre bene </td><td> Evergreen scientifico </td><td> 222-8044-01F </td></tr><tr><td> SuperFrost / Plus Microscopio Slides </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4 ml fiale chiaro </td><td> Scientifico Nazionale </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

La creazione di una rete funzionale vaso sanguigno è una parte essenziale di organogenesi, ed è necessario per il funzionamento ottimale degli organi. Per esempio, nella formazione timo corretta vascolarizzazione ed il modello è indispensabile per l&#39;ingresso timocita nell&#39;organo e maturo delle cellule T uscita alla periferia. La disposizione spaziale dei vasi sanguigni nel timo dipende segnali provenienti dal microambiente locale, cioè le cellule epiteliali timiche (TEC). Diversi rapporti recenti suggeriscono che la perturbazione di questi risultati segnali in timo vaso sanguigno 1,2 difetti. Studi precedenti hanno descritto le tecniche utilizzate per etichettare il neonatale e adulto vascolarizzazione del timo 1,2. Si dimostra qui una tecnica per l&#39;etichettatura dei vasi sanguigni nel timo embrionale. Questo metodo combina l&#39;uso di FITC-destrano o Griffonia (Bandeiraea) Simplicifolia lectina I (GSL 1 - isolectin B 4) iniezioni vena facciale e CD31 colorazione anticorpi per identificare timo strutture vascolari e PDGFR-β per etichettare timo mesenchima perivascolare 3-5. La possibilità di utilizzare criosezioni o sezioni vibratome inoltre è fornita. Questo protocollo può essere utilizzato per identificare timo difetti vascolari, che è fondamentale per definire i ruoli di TEC-molecole derivate timo nella formazione dei vasi sanguigni. Come metodo di etichette intero sistema vascolare, può anche essere usato per analizzare le reti vascolari in molti organi e tessuti in tutto l&#39;embrione tra pelle e cuore 6-10.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

Questo articolo descrive un metodo per l&#39;etichettatura di pelle embrionale e vasi sanguigni timo.

Un metodo per l&#39;etichettatura vasi embrionali nei topi

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

a-method-for-labeling-vasculature-in-embryonic-mice

Research

JoVE Journal

Immunology and Infection

15.5K Views.  University of Georgia. Questo articolo descrive un metodo per l'etichettatura di pelle embrionale e vasi sanguigni timo.

Video: A Method for Labeling Vasculature in Embryonic Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Ricerca

Immunologia e infezioni

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferenza genotipica del virus HIV-1 Tropismo utilizzo basati sulla popolazione sequenziamento del V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non invasiva di imaging ottico del sistema vascolare linfatico di un mouse

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D imaging multimodale di<em&gt; Citrobacter rodentium</em&gt; Infezioni in topi

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induzione di paralisi e di lesioni del sistema visivo in topi da specifiche cellule T per neuromielite ottica Autoantigen acquaporina-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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 metodo per la misurazione della funzione della ghiandola salivaria in topi

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

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Trombosi venosa profonda indotta dalla stasi in topi monitorati dall'ecografia ad alta frequenza

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Un metodo delle nanoparticelle d'argento per il miglioramento di sindrome di Atresia biliare nei topi

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Methods Article

Un metodo per l'etichettatura vasi embrionali nei topi