Diese Arbeit wurde von den Zuwendungs-Zahlen R01AI055001 und R01AI082127 von NIAID zur NRM und SREB Dissertation Fellowship Award JLB unterstützt.

1. Fluorescein markiertem Dextran und GSL I-isolectin B 4 V. facialis Injektionen zu embryonalen Blutgefäßen Label <ol><li> Bereiten FITC-Dextran (50ug/mL) in Phosphat-gepufferter Saline (PBS) oder GSL 1 - isolectin B 4 (20ug/200uL) in PBS in einem 1,5 ml Eppendorf-Röhrchen und warm bis 37 ° C. Add 100 ul auf Lager 1,25 Fast Green / PBS, die FITC-Dextran-Lösung (Gesamtvolumen 1 ml) und 180uL of stock 1,25 Fast Green / PBS auf die GSL 1 - isolectin B 4 (Gesamtvolumen 200 &amp;mgr; l), so dass die Lösung sichtlich blau. </li><li> Dissect E14.5-E18.5 Embryonen und Dottersack zusammen, so dass die Allantois Stiel (Nabel-Arterie und Vene) intakt. </li><li> -Embryonen eine neue Petrie Schüssel (60 X 15 mm) und tauche sie in PBS bei Raumtemperatur. </li><li> Position des Embryos zu einer sagittalen Ansicht des Kopf / Gesicht geben. Verwenden Sie Micro-Pinzette vorsichtig fassen den Embryo an der Spitze. </li><li> Mit einer 30G Nadel injiziert 50ul FITC-Dextran (50ug/mL) oder GSL 1 - isolectin B 4 (20ug in 200 &amp;mgr; l PBS) in die V. facialis und dabei die Nadel in Richtung der Rückseite des Kopfes. </li><li> Wenn der Farbstoff sichtbar in die Nabelvene ist, entfernen Sie die Nadel und trennen Sie die Embryos aus der Allantois-Stiel (Nabel-Arterie und Vene). </li><li> Nach Injektionen, erlauben Embryo in PBS bei Raumtemperatur bleibt für 2-3 Minuten, so dass der Farbstoff zirkuliert im gesamten Embryo. </li></ol> 2. Whole-mount Analyse der Haut Gefäßsystem <ol><li> Nach Abzug der Farbstoff im gesamten Embryo zirkulieren, zu entfernen Hautproben aus Regionen der Extremitäten, Rücken und Bauch, etc. 8,9. </li><li> Waschen Sie die Haut Probe in kaltem PBS, und fixieren in 4% PFA / PBS für 2 Stunden 8,9. 3 x waschen für jeweils 10 Minuten in 4 ml klare Flasche mit 2 ml kaltem PBS. </li><li> Legen Sie Hautprobe auf einen Objektträger und 100 ul der Montage Medien zu jeder Folie und einem Deckglas. </li><li> Lassen Sie die Objektträger in einem dunklen Lagerraum trocken. </li><li> Fahren Sie mit 2 &#39;Image Acquisition &quot;Abschnitt Schritt. </li></ol> 3. Multi-Farb-Kennzeichnung von Thymus und Herz Gefäßsystem und perivaskulären Zellen für Gefrierschnitte (aus Abschnitt 1, Schritt 7 weiter) <ol><li> &#39;Flash freeze&#39; ganze Embryo in flüssigem Stickstoff. Embryonen werden können und bei -80 ° C bis zur Analyse. </li><li> Alternativ herauspräparieren Thymus, spülen in 4 ° C PBS, und fix in 2 ml 4% Paraformaldehyd (PFA) / PBS für 2 Stunden. 3 x je 10 Minuten in kaltem PBS, Ort Thymi in OCT und einfrieren und lagern bis zur Verwendung bei -80 ° C. </li><li> Für Kryoschneiden verbreitete ÜLG auf einen Abschnitt &#39;block&#39; und montieren Sie den Embryo oder seziert Organe / Gewebe zum Schneiden. </li><li> Cut gefrorenen Gewebe in 10 pm dicke Schnitte und sammeln auf Folien. </li><li> Fix Abschnitte in Aceton für 5-10 Minuten. 3 x waschen mit kaltem TBS. </li><li> Block in 10% Esel Serum / TBS in einer feuchten Kammer bei Raumtemperatur. </li><li> Inkubieren Abschnitte 1 Stunde Übernachtung mit 100 ul Primärantikörper in einer feuchten Kammer bei 4 ° C: in diesem Beispiel verwenden wir Ratte anti-Maus-CD31 (1:100) zu kennzeichnen Endothel und Ziege anti-Maus PDGFR-β (1:100) zu kennzeichnen perivaskulären Zellen. Es ist sinnvoll, Folien mit individuell zugeschnittenen Streifen Parafilm abdecken, um sicherzustellen, dass der Antikörper gleichmäßig über den Querschnitt verteilt. </li><li> Nach der Inkubation mit dem Primärantikörper, waschen Abschnitte 3 mal in kaltem TBS. Inkubieren mit 100 ul entsprechenden Sekundärantikörper für mindestens 30 Minuten. </li><li> 3 x waschen mit kaltem TBS. 100 l der Montage Medien zu jeder Folie und einem Deckglas. </li><li> Lassen Sie die Objektträger in einem dunklen Lagerraum trocken. </li><li> Proceed to &#39;Image Acquisition &quot;Abschnitt. </li></ol> 4. Multi-Farb-Kennzeichnung von Thymus Gefäßsystem und perivaskulären Zellen für Vibratom Abschnitte (aus Abschnitt 1, Schritt 7 weiter) <ol><li> Dissect aus Thymus Lappen vom Embryo und spülen Sie mit kaltem PBS. </li><li> Fix Thymus in 4% PFA / PBS bei Raumtemperatur für 2 Stunden. </li><li> Wash in PBS-Triton X (0,15%) 3-mal, 10 Minuten und Platz Thymi in einer kleinen Kunststoff-Kartusche und tauchen in 4% niedrig schmelzende Agarose / PBS (~ 4 ° C). Der Thymus ist in Kontakt mit der Unterseite der Patrone sein. </li><li> Lassen Agarose auf Eis (3-5 Minuten) zu festigen. Verwenden einer Rasierklinge abgeschnitten überschüssige Agarose. Fügen Sie Leim auf die Vibratom Block und halten Probe, um den Block. </li><li> Add kaltem PBS zu Vibratom Wasserbad, bis die Probe und Klinge eingetaucht sind. </li><li> Set Geschwindigkeit und Amplitude (hoher Amplitude und niedriger mittlerer Geschwindigkeit ist ideal für weiche Thymus Abschnitte). Die Amplitude reduziert werden, wenn Teile zerbrechen aufgrund von zu viel Aufregung sein. </li><li> Cut 50 um Abschnitte. </li><li> Mit einem Pinsel, sammeln Abschnitte in einem 24-well Mikroplatten in kaltem PBS. </li><li> Block Abschnitte in 500 ul 10% Esel-Serum in PBS-Triton X (0,15%) für 30 Minuten. </li><li> Inkubieren Abschnitte für 8 Stunden bis über Nacht mit dem primären Antikörper, wie anti-CD31-und Anti-PDGFR-β, In einer überdachten 24-Well-Mikroplatten. </li><li> 3 x waschen mit PBS-Triton X (0,15%) auf insgesamt 8 Stunden bei 4 ° C. </li><li> Block Abschnitte in 10% Esel-Serum in PBS-Triton X (0,15%) für 30 Minuten. </li><li> Inkubieren Abschnitte für 8 Stunden bis über Nacht bei 4 ° C mit entsprechenden Sekundärantikörper. </li><li> 3 x waschen mit PBS-Triton X (0,15%) auf insgesamt 8 Stunden bei 4 ° C. </li><li> Re-fix Proben in 4% PFA / PBS für 30 Minuten auf Eis. </li><li> 3 x waschen mit PBS-Triton X (0,15%) über 30 Minuten auf Eis. </li><li> Entwässern Proben durch ein abgestuftes MeOH / PBS-Triton X-Serie: 25% MeOH, 50% MeOH, 75% MeOH und 100% MeOH in 10 Minuten für jeden Schritt. Ersetzen Sie 100% MeOH mit frischem MeOH nach 10 Minuten und inkubieren für 1 Stunde bei Raumtemperatur. </li><li> In einem Glasbehälter, mischen BABB (Benzylalkohol: Benzylbenzoat) im Verhältnis 1:2. Kombinieren Sie BABB mit MeOH für eine Endkonzentration von 50% BABB und 50% MeOH. Inkubieren Sie die Probe in BABB: MeOH für 10-15 Minuten. </li><li> Transfer-Probe auf einem Glasbehälter mit 100% BABB und Inkubation für 10-15 Minuten oder bis gelöscht, bei Raumtemperatur. </li><li> Füllen Depression slide (0.7mm Tiefe) mit frischem 100% BABB und Transfer Probe auf die Seite. Add Deckglas (Nr. 1,5) und Dichtung mit 2-3 Schichten von Nagellack. Lassen Sie Nagellack zu härten im Dunkeln bei Raumtemperatur, dann speichern Probe bei 4 ° C. Hinweis: Folien müssen absolut dicht vor dem konfokalen Bildaufnahme werden. Die Bilder sollten innerhalb von 12-24 Stunden erworben werden, als Fluoreszenzfarbstoffe in BABB verblassen kann. </li><li> Proceed to &#39;Image Acquisition &quot;Abschnitt. </li></ol> 5. Bildaufnahme <ol><li> Bild 10 um Gefrierschnitten mit einem konfokalen Mikroskop mit dem Plan-Apochromat 20X/0.8 Ziel (512 x 512 Pixel) mit 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 - und 633-nm-Laser-Linien. </li><li> Erwerben Sie konfokalen z-Abschnitte whole mount Haut und 50 um Agarose eingebetteten Schnitten mit der Plan-Apochromat 10X/0.4 Ziel (512 x 512 Pixel) mit 488 - (FITC-dextran/GSL 1 - isolectin B 4), 543 -, und 633-nm-Laser-Linien. Serielle Z-Abschnitte sollten nacheinander auf 1-Mikron gesammelt werden für den jeweiligen Kanal. </li><li> Rekonstruieren seriellen Z-Sektionen mit Zeiss AxioVision 4.6 oder andere Bild-Analyse-Software. </li></ol> 6. Repräsentative Ergebnisse: Effiziente Kennzeichnung des embryonalen Gefäßsystems ist entscheidend für die Beurteilung von Defekten an Blutgefässen in embryonalen Mäuse. Abbildung 1 zeigt spezifische Kennzeichnung von E16.5 Thymus Blutgefäße (1A-B) und Co-Markierung mit CD31 (1B), zusätzlich zu Verfärbung der rechten und linken Ventrikel (1E-F), bzw.. Die GSL I-isolectin B-4-Protokoll für Gefrierschnitte wie in den Abschnitten 1, 3 und 5 beschrieben wurde in diesen Experimenten verwendet. Whole-mount Kennzeichnung der Haut Blutgefäß-Netz auf E16.5 Mäusen, mit Hilfe der Protokolle in den Abschnitten 1, 2 beschrieben, und 5 ist in Abbildung 1C-D gezeigt. <img alt="Abbildung 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> Abbildung 1 Legend. FITC GSL I - isolectin B 4 V. facialis Injektionen in E16.5 Maus-Embryonen. a. Gefrier der embryonalen Thymus nach der Injektion. b. Merge von CD31 co-Kennzeichnung mit isolectin B 4. c. und d. Whole-mount der embryonalen Haut Gefäße nach der Injektion. e. und f. Gefrier von embryonalen Herzen e. (rechts Ventrikel) f. (linker Ventrikel) nach der Injektion.

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Keine Interessenskonflikte erklärt.

Whole-mount und PECAM-1 (CD31) Flecken auf Abschnitte sind den herkömmlichen Verfahren zur Kennzeichnung der Gefäße in der embryonalen Mäuse. Diese Methoden erfordern den Einsatz von direkten und / oder indirekte Immunfluoreszenz und Reinigungsmittel zur Mäusegewebe permeabilisieren. Dies erweist sich als recht zeitnah zu verarbeiten. Hier haben wir eingesetzten FITC-Dextran oder isolectin B 4 V. facialis Injektionen direkt beschriften Sie die embryonale Gefäßsystem, wodurch die Voraussetzung für Anti...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> FITC-Dextran </td><td> Sigma </td><td> FD150S-1G </td></tr><tr><td> Fluorescein GSL 1 - isolectin B 4 </td><td> Vector Laboratories </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 Biologicals </td><td> S11550 </td></tr><tr><td> Optimal Cutting Temperature Compound (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> Aceton </td><td> JT Baker </td><td> 9006-33 </td></tr><tr><td> Donkey Serum </td><td> Jackson </td><td> 017-000-121 </td></tr><tr><td> Ratte anti-Maus-CD31, </td><td> BD Pharmingen </td><td> 558736 </td></tr><tr><td> Ziege anti-Maus PDGFR-β </td><td> R &amp; D Systems </td><td> AF1042 </td></tr><tr><td> Esel-anti-Ratte CD31 Alexa 647 (Invitrogen) </td><td> Biolegend </td><td> 102516 </td></tr><tr><td> Esel anti-Ziege 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> Low Melt Agarose / PBS </td><td> Sigma-Aldrich </td><td> A9414-25G </td></tr><tr><td> Methanol </td><td> Fisher Scientific </td><td> A413-4 </td></tr><tr><td> Benzylalkohol </td><td> Acros Scientific </td><td> 148390010 </td></tr><tr><td> Benzyl Benzoate </td><td> Acros Scientific </td><td> 105860010 </td></tr><tr><td> Depression rutscht </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> Deckglas (22x22) -1,5 </td><td> Thermo Scientific </td><td> 152222 </td></tr><tr><td> Zeiss LSM 510 Meta konfokalen Mikroskop </td><td> Zeiss </td><td></td></tr><tr><td> Micro Pinzette </td><td> Roboz </td><td> RS-5135 </td></tr><tr><td> Parafilm Nr. OM992 </td><td> Fisher Scientific </td><td> 13-374-16 </td></tr><tr><td> 12 und 24 Well-Mikroplatten </td><td> Evergreen Scientific </td><td> 222-8044-01F </td></tr><tr><td> Superfrost / Plus Mikropräparate </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> 4mL klaren Fläschchen </td><td> National Scientific </td><td> B7800-2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

Die Einrichtung eines funktionalen Blutgefäß-Netzwerk ist ein wesentlicher Bestandteil der Organogenese und ist für eine optimale Organfunktion erforderlich. Zum Beispiel in der Thymusdrüse richtigen Gefäße Bildung und Strukturierung ist wesentlich für Thymozyten Einstieg in die Orgel und reifen T-Zellen-Ausgang an die Peripherie. Die räumliche Anordnung der Blutgefäße in der Thymusdrüse ist abhängig von Signalen aus dem lokalen Mikroumgebung, nämlich Thymus Epithelzellen (TEC). Mehrere neuere Berichte deuten darauf hin, dass eine Störung dieser Signale führt in Thymus Blutgefäß Defekte 1,2. Frühere Studien haben Techniken verwendet, um die Neugeborenen und adulten Thymus Gefäßsystem 1,2 label beschrieben. Wir zeigen hier eine Technik zur Kennzeichnung Blutgefäße in der embryonalen Thymus. Diese Methode kombiniert die Verwendung von FITC-Dextran oder Griffonia (Bandeiraea) Simplicifolia Lektin I (GSL 1 - isolectin B 4) V. facialis Injektionen und CD31 Antikörper-Färbung zu Thymus vaskulären Strukturen und PDGFR-β zu Thymus perivaskulären Mesenchym 3-5 Etikett zu kennzeichnen. Die Möglichkeit der Verwendung Kryoschnitten oder Vibratom Abschnitte ist ebenfalls vorhanden. Dieses Protokoll kann verwendet werden, um Thymus vaskulären Mängel, die entscheidend für die Festlegung der Rolle der TEC-basierten Molekülen in Thymus die Bildung von Blutgefäßen zu identifizieren. Da die Methode kennzeichnet die gesamte Gefäßsystem, kann es auch verwendet, um den vaskulären Netze in mehreren Organen und Geweben während der Embryo einschließlich Haut-und Herz 6-10 analysieren.

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

Dieser Artikel beschreibt eine Methode zur Kennzeichnung embryonalen Haut und Thymus Blutgefäße.

Ein Verfahren zur Markierung Gefäßsystem in embryonalen Mäuse

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 Aufrufe.  University of Georgia. Dieser Artikel beschreibt eine Methode zur Kennzeichnung embryonalen Haut und Thymus Blutgefäße.

Serie: 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.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

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

Forschung

Immunologie und Infektion

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.

Genotypische Inference von HIV-1 Tropismus mit Populations-basierte Sequenzierung von 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.

Visualizing Dengue Virus durch 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 ...

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.

Nicht-invasive optische Abbildung des lymphatischen Gefäßsystem eines Maus

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 von Multimodalität<em&gt; Citrobacter rodentium</em&gt; Infektionen bei Mäusen

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.

Ein Nicht-invasive und in technischer Hinsicht nicht intensive Verfahren zur Induktion und Phänotypisierung für experimentelle bakterielle Pneumonie bei Mäusen

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.

Induktion von Lähmung und visuelles System Verletzungen bei Mäusen durch T-Zellen spezifisch für 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 ...

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.

Eine Methode zur Messung der Speicheldrüse Funktion bei Mäusen

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.

Tiefe Venenthrombose, induziert durch Stauung in den Mäusen durch Hochfrequenz-Sonographie überwacht

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.

Eine Silber Nanopartikel-Methode für bessernde biliären Atresie-Syndrom bei Mäusen

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 ...