Bu eser hibe numaraları R01AI055001 ve NIAID gelen UYM ve JLB için SREB Tezi Bursu Ödülü R01AI082127 tarafından desteklenmiştir.

1. Flöresein dekstran ve GSL I-isolectin B 4 yüz ven enjeksiyonlar embriyonik damar etiketlemek için etiketlenmiş <ol><li> Fosfat içinde FITC-dekstran (50ug/mL) hazırlayın Tamponlu Tuz (PBS) veya GSL 1 - isolectin B, bir 1,5 mL Eppendorf tüpü içinde PBS içinde 4 (20ug/200uL) ve 37 ° C&#39;de sıcak Yeşil / PBS GSL 1. Yeşil / PBS FITC-Dekstran solüsyonunun (toplam hacmi 1 ml) ve stok 1.25mm Fast 180uL stok 1.25mm Fast 100ul Ekle - isolectin çözümdür böylece B 4 (toplam hacim 200ul), gözle görülür mavi. </li><li> Allantoik sapı (umblikal arter ve ven) zedelemeden, birlikte E14.5-E18.5 embriyo ve yolk kesesi teşrih. </li><li> Yeni bir Petrie tabak (60 X 15 mm) kadar embriyo transferi ve oda sıcaklığında PBS içinde sokmaktan. </li><li> Baş / yüzün sagittal bir görünüm sağlamak için embriyo yerleştirin. Yavaşça başında embriyo kavramak için mikro diseksiyon forseps kullanın. </li><li> Bir 30G iğne kullanarak, 50uL FITC-dekstran (50ug/mL) veya GSL enjekte1 - isolectin başın arkasına doğru iğne işaret fasiyal vene B 4 (200ul PBS 20ug). </li><li> Boya umbilikal ven görünür olduğunda, iğne kaldırmak ve allantoik sapı (umbilikal arter ve ven) gelen embriyo ayırın. </li><li> Enjeksiyonu takiben, embriyo boya dolaşır embriyo böylece 2-3 dakika için oda sıcaklığında PBS içinde kalmasını sağlamaktadır. </li></ol> 2. Deri vaskülatürü Tüm montaj analizi <ol><li> Boya embriyo boyunca dolaşmaya izin verdikten sonra, sırt, bacakların bölgelerinden alınan deri örnekleri kaldırmak ve mide, vb 8,9. </li><li> Soğuk PBS içinde deri örneği yıkayın ve 2 saat 8,9 için% 4 PFA / PBS düzeltin. 10 dakika 2mL soğuk PBS ile 4 mL berrak flakonda her biri için 3 kere yıkayın. </li><li> Bir mikroskop yerleştirin deri örneği her slayt ve kapak cam montaj medya 100 ul kaydırın ve ekleyin. </li><li> Slaytlar karanlık bir depolama alanında kurumasını bekleyin. </li><li> P2 &#39;Image Acquisition&#39; bölümüne Adım roceed. </li></ol> 3. Timus ve kalp damar ve cryosections için perivasküler hücrelerin Çok renkli etiketleme (Bölüm 1, Adım 7 Devam) <ol><li> Sıvı azot &#39;Flaş don&#39; bütün embriyo. Embriyolar olması ve -80 ° C&#39;de analize kadar saklanır olabilir. </li><li> Alternatif olarak, timus dışarı incelemek 4 ° C&#39;de PBS ile yıkanır ve 2 saat boyunca 2mL% 4 paraformaldehit (PFA) / PBS içinde çözmek. -80 Kullanıma kadar soğuk PBS, OCT yer thymi ve dondurma ve mağaza ile 10 dakika boyunca 3 kez yıkayın ° C </li><li> Cryosectioning için bir bölüm &#39;blok&#39; üzerinde Ekim yaymak ve kesit için embriyo veya disseke organ / dokuların monte. </li><li> 10 mikron kalınlığında kesitler halinde dondurulmuş doku Kes ve slaytlar üzerinde toplamak. </li><li> 5-10 dakika aseton bölümler düzeltildi. Soğuk TBS içinde 3 kere yıkayın. </li><li> Oda sıcaklığında bir nem odası içinde% 10 eşek serum / TBS içinde engelleme. </li><li> Primer 100 ul ile 1 saatlik gecede için bölümler inkübe4 bir nem odası antikorlar ° C: Bu örnekte, biz etiket endotele Sıçan anti-fare CD31 (1:100) ve keçi anti-fare perivasküler hücreler etiketlemek için PDGFR-β (1:100) kullanın. Bu antikor düzgün bölümüne yayılmış olduğundan emin olmak için ayrı ayrı kesilmiş Parafilm şeritler ile slaytlar karşılamak için yararlıdır. </li><li> Primer antikor ile inkübasyondan sonra, soğuk TBS bölümler 3 kere yıkayın. En az 30 dakika için uygun sekonder antikor 100 ul ile inkübe edin. </li><li> Soğuk TBS içinde 3 kere yıkayın. Her slayt ve kapak cam montaj medya 100 ul ekleyin. </li><li> Slaytlar karanlık bir depolama alanında kurumasını bekleyin. </li><li> &#39;Image Acquisition&#39; bölümüne geçin. </li></ol> 4. Timus ve damarsal vibratome bölümleri için perivasküler hücrelerin Çok renkli etiketleme (Bölüm 1, Adım 7 Devam) <ol><li> Embriyodan timus lobları dışarı inceleyin ve soğuk PBS içinde yıkayın. </li><li> 2 saat için oda sıcaklığında% 4 PFA / PBS içinde timus saptamaks. </li><li> PBS-Triton X (0.15%) 3 kez, 10 dakika ve erime düşük 4% küçük bir plastik kartuş ve batığın yerini thymi yıkayın agaroz / PBS (~ 4 ° C). Timus kartuşun dibi ile temas halinde olmalıdır. </li><li> Agaroz buz (3-5 dakika) katılaşmaya bırakın. Aşırı agaroz kesmek için jilet kullanın. Vibratome blok tutkal ekleyin ve blok numune uygun. </li><li> Örnek ve bıçak batırılır kadar vibratome su banyosuna soğuk PBS ekleyin. </li><li> Hız ve genlik (yüksek genlikli ve düşük-orta hızda yumuşak timus bölümleri için idealdir) ayarlayın. Bölümlerde aşırı ajitasyon nedeniyle break up genliği azaltılmalıdır. </li><li> 50 um bölümleri kesiniz. </li><li> Bir fırça kullanarak, soğuk PBS içinde 24-kuyu mikroplak bölümler toplamak. </li><li> 30 dakika boyunca PBS-Triton X (% 0.15) içinde% 10 eşek serum ve 500 ul içinde blok bölümleri. </li><li> Bu tür anti-CD31 ve anti-PDGFR-β gibi birincil antikor ile bir gece 8 saat için bölümler için, inkübe, Kapalı bir 24-çukurlu bir mikrolevhadaki. </li><li> 4 ° C&#39;de 8 saatlik bir toplam boyunca PBS-Triton X (% 0.15) içinde 3 kere yıkayın </li><li> 30 dakika boyunca PBS-Triton X (% 0.15) içinde% 10 eşek serum içinde blok bölümleri. </li><li> 8 saat bölümler inkübe 4 gecede ° C uygun ikincil antikor ile. </li><li> 4 ° C&#39;de 8 saatlik bir toplam boyunca PBS-Triton X (% 0.15) içinde 3 kere yıkayın </li><li> Re-fix örneklerinde% 4 PFA / buz üzerinde 30 dakika süreyle PBS. </li><li> Buz üzerinde 30 dakika PBS-Triton X (0.15%) 3 kez yıkayın. </li><li> % 25 MeOH,% 50 MeOH,% 75 MeOH, ve her adım için 10 dakika sonra% 100 MeOH: bir aşamalı MeOH / PBS-Triton X dizi örnekleri kurutmak. 10 dakika sonra taze MeOH ile% 100 MeOH değiştirin ve oda sıcaklığında 1 saat süreyle inkübe edilir. </li><li> Bir cam kapta, mix Babb (Benzil Alkol: Benzil Benzoat) 1:2 oranında. Babb% 50 ve% 50 MeOH bir nihai konsantrasyon için MeOH ile Babb birleştirin. 10-15 dakika MeOH: Babb örnek inkübe edin. </li><li> Transferi sa10-15 dakika için% 100 Babb ve inkübe ile bir cam kap için mple veya kadar oda sıcaklığında, temizlenir. </li><li> Tarafı taze% 100 Babb ve transfer örneği ile depresyon slayt (0.7mm derinlik) doldurun. Cam (No 1.5) kapak ve oje 2-3 kat ile mühür ekleyin. Oda sıcaklığında karanlıkta sertleşmesine oje bırakın ve daha sonra 4 ° C&#39;de örnek saklamak Not: Slaytlar tamamen konfokal görüntü alımı öncesinde kapatılmalıdır. Floresan boyalar Babb fade gibi Görüntüler, 12-24 saat içinde satın alınmalıdır. </li><li> &#39;Image Acquisition&#39; bölümüne geçin. </li></ol> 5. Görüntü edinimi <ol><li> Görüntü 10 488 ile Plan-Apochromat 20X/0.8 nesnel (512 X 512 piksel) kullanarak bir konfokal mikroskop ile mikron frozen - (FITC-dextran/GSL 1 - B 4 isolectin), 543 - ve 633-nm lazer çizgiler. </li><li> Plan-Apochroma kullanarak bütün montaj cilt ve 50 mikron agaroz-gömülü kesitlerin konfokal z bölümler Edinme<html> t 10X/0.4 488 ile nesnel (512 X 512 piksel) - (FITC-dextran/GSL 1 - B 4 isolectin), 543 - ve 633-nm lazer çizgiler. Seri Z bölümleri ilgili her kanal için 1 mikron sıralı olarak toplanmalıdır. </li><li> Seri Yeniden İnşa Zeiss Axiovision 4.6 ya da diğer görüntü analiz yazılımı kullanılarak Z-bölümler. </li></ol> 6. Temsilcisi Sonuçlar: Embriyonik damar Verimli etiketleme embriyonik farelerde kan damarı kusurları değerlendirmek için önemlidir. Şekil 1, sırasıyla sağ ve sol ventrikül (1E-F) arasında boyama ek olarak, özel E16.5 timüs kan damarlarının etiketleme (1A-B) ve CD31 (1B) ile birlikte etiketleme, göstermektedir. Gibi bölümlere 1, 3 içinde belirtilen cryosections ve 5 için GSL I-isolectin B 4 protokol bu deneylerde kullanılmıştır. Bütün monte edilir, E16.5 fareler üzerinde cilt kan damar ağının bölümleri etiketleme 1, 2 içerisinde tarif edilen protokoller kullanılarak, ve 5, şekil 1C-B &#39;de gösterilmiştir. ntent &quot;&gt; <img alt="Şekil 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> 1. Legend Şekil. FITC GSL I - E16.5 fare embriyolarına isolectin B 4 yüz ven enjeksiyonlar. embriyonik kalp e enjeksiyonu takiben embriyonik Timus bir. Cryosection. b. isolectin B 4 CD31 ko-etiketleme Birleştirme. c. ve d. embriyonik deri vaskülatürü aşağıdaki enjeksiyon Tüm montaj. e. ve f. Cryosection. (sağ ventrikül) f. (sol ventrikül) aşağıdaki enjeksiyon.

<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>

Çıkar çatışması ilan etti.

Bölümlerde Tüm-mount ve PECAM-1 (CD31) boyama embriyonik farelerde damar etiketleme için geleneksel yöntemler vardır. Bu yöntemler doğrudan ve / veya dolaylı immunfloresan ve deterjan kullanımı fare doku geçirgenliği gerektirir. Bu oldukça zamanında süreç olduğunu kanıtlıyor. Burada, FITC-dekstran ya isolectin B 4 yüz damar enjeksiyonu doğrudan böylece antikor etiketleme adımlar için ihtiyacı ortadan kaldırarak, embriyonik damar etiketlemek için istihdam var. &#39;Sızdıran&#39; f...

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> FITC-dekstran </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Flöresein GSL 1 etiketli - B 4 isolectin </td><td> Vektör Laboratuvarları </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> Fetal Bovine Serum </td><td> Atlanta Biyolojik </td><td> S11550 </td></tr><tr><td> Optimal Kesme Sıcaklık Bileşik (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> Aseton </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Eşek Serum </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> Sıçan anti-fare CD31, </td><td> BD Pharmingen </td><td> 558736 </td></tr><tr><td> keçi anti-fare PDGFR-β </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> eşek anti-fare CD31 Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> eşek anti-keçi 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> Düşük erime agaroz / PBS </td><td> Sigma-Aldrich </td><td> A9414-25G </td></tr><tr><td> Metanol </td><td> Fisher Scientific </td><td> A413-4 </td></tr><tr><td> Benzil Alkol </td><td> Acros Bilimsel </td><td> 148390010 </td></tr><tr><td> Benzil Benzoat </td><td> Acros Bilimsel </td><td> 105860010 </td></tr><tr><td> Depresyon slaytlar </td><td> Fisher Scientific </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> Elektron Mikroskopi Bilimleri </td><td> 17985-10 </td></tr><tr><td> Cam (22x22) -1.5 Kapak </td><td> Termo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Meta Konfokal Mikroskop </td><td> Zeiss </td><td></td></tr><tr><td> Mikro diseksiyon forseps </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 ve 24 de mikroplaklar </td><td> Evergreen Bilimsel </td><td> 222-8044-01F </td></tr><tr><td> Superfrost / Artı Mikroskop Slaytlar </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4 mL berrak şişeleri </td><td> Ulusal Bilimsel </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

Fonksiyonel bir damar ağının kurulması organogenez önemli bir parçası olduğunu ve optimal organ fonksiyonları için gereklidir. Örneğin, timüs uygun damar oluşumu ve dokusunda organ ve çevre için olgun T-hücresi çıkışında içine timosit giriş için gereklidir. Timus kan damarlarının mekansal düzenleme yerel mikroçevrede, yani timik epitel hücreleri (TEC) gelen sinyalleri bağlıdır. Birkaç yeni raporlar timus damar kusurları 1,2 bu sinyallerin sonuçları bu bozulma göstermektedir. Önceki çalışmalarda yenidoğan ve yetişkin timus damar 1,2 etiketlemek için kullanılan teknikler tarif var. Biz burada embriyonik timus kan damarlarının etiketlenmesine yönelik bir tekniği göstermek. Yüz damar enjeksiyonu ve CD31 antikor boyama timüs va tespit etmek - bu yöntem simplicifolia lektin I (isolectin B 4 GSL 1) FITC-dekstran ya da Griffonia (Bandeiraea) kullanılması birleştirirscular yapıları ve PDGFR-β timik perivasküler mezenşimi 3-5 etiketlemek için. Cryosections veya vibratome bölümleri kullanma seçeneği de sağlanır. Bu protokol, timüs kan damarı oluşumuna TEC-türetilen molekülleri rollerin tanımlanması için kritik olan timus damar bozuklukları, belirlemek için kullanılabilir. Yöntemi, tüm damar etiket olarak, aynı zamanda deri ve kalp 6-10 içeren embriyo boyunca birden fazla organ ve dokularda damar ağları analiz etmek için de kullanılabilir.

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

Bu makale, embriyonik deri ve timüs kan damarlarının etiketlenmesine yönelik bir yöntem açıklanmaktadır.

Embriyonik Farelerde damarları Etiketleme Bir Yöntem

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 Görüntüleme.  University of Georgia. Bu makale, embriyonik deri ve timüs kan damarlarının etiketlenmesine yönelik bir yöntem açıklanmaktadır.

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.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

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

İmmünoloji ve Enfeksiyon

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.

HIV-1 tropizm genotipik Çıkarım kullanarak Nüfus tabanlı Sıralama 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.

Alexa Fluor Etiketleme Dang Virüs görselleştirme

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.

Bir Mouse Lenfatik damarları Non-invaziv Optik Görüntüleme

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 modelli Görüntüleme<em&gt; Citrobacter rodentium</emFarelerde&gt; Enfeksiyonlar

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.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

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.

Felç ve görsel sistem yaralanma tarafından Nöromyelitis Optica Autoantigen Aquaporin-4 T hücreleri belirli farelerde indüksiyon

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.

Tükürük bezi işlevi farelerde ölçülmesi için bir yöntem

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.

Derin Ven Trombozu Staz farelerde yüksek frekans Ultrasonografi tarafından izlenen tarafından indüklenen

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.

Safra atrezi sendromu farelerde Ameliorating için gümüş bir nanopartikül yöntemi

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

Embriyonik Farelerde damarları Etiketleme Bir Yöntem