This work was supported by grant numbers R01AI055001 and R01AI082127 from NIAID to NRM and SREB Dissertation Fellowship Award to JLB.

1. Fluorescein labeled dextran and GSL I-isolectin B4 facial vein injections to label embryonic vasculature
<ol>
	<li>Prepare FITC-dextran (50ug/mL) in Phosphate Buffered Saline (PBS) or GSL 1 - isolectin B4 (20ug/200uL) in PBS in a 1.5mL Eppendorf tube and warm to 37&deg;C. Add 100uL of stock 1.25mM Fast Green/PBS to the FITC-dextran solution (total volume 1mL) and 180uL of stock 1.25mM Fast Green/PBS to the GSL 1 - isolectin B4 (total volume 200uL), so that the solution is visibly blue.</li>
	<li>Dissect E14.5-E18.5 embryos and yolk sac together, leaving the allantoic stalk (umbilical artery and vein) intact.</li>
	<li>Transfer embryos to a new petrie dish (60 X 15 mm) and immerse them in PBS at room temperature.</li>
	<li>Position the embryo to provide a sagittal view of the head/face. Use micro dissecting forceps to gently grasp the embryo at the head.</li>
	<li>Using a 30G needle, inject 50uL FITC-dextran (50ug/mL) or GSL 1 - isolectin B4 (20ug in 200uL PBS) into the facial vein pointing the needle toward the back of the head.</li>
	<li>When the dye is visible in the umbilical vein, remove the needle and separate the embryo from the allantoic stalk (umbilical artery and vein).</li>
	<li>Following injections, allow embryo to remain in PBS at room temperature for 2-3 minutes so that the dye circulates throughout the embryo.</li>
</ol>
2. Whole-mount analysis of skin vasculature
<ol>
	<li>After allowing the dye to circulate throughout the embryo, remove skin samples from regions of the limbs, back, and stomach, etc. 8,9.</li>
	<li>Wash skin sample in cold PBS, and fix in 4% PFA/PBS for 2 hours 8,9. Wash 3 times for 10 minutes each in 4mL clear vial with 2mL cold PBS.</li>
	<li>Place skin sample on a microscope slide and add 100 &mu;l of mounting media to each slide and a cover glass.</li>
	<li>Allow slides to dry in a dark storage area.</li>
	<li>Proceed to Step 2 'Image Acquisition' section.</li>
</ol>
3. Multi-color labeling of thymus and heart vasculature and perivascular cells for cryosections (Continue from Section 1, Step 7)
<ol>
	<li>'Flash freeze' whole embryo in liquid nitrogen. Embryos can be and stored at -80&deg;C until analysis.</li>
	<li>Alternatively, dissect out thymus, rinse in 4&deg;C PBS, and fix in 2mL 4% Paraformaldehyde (PFA)/PBS for 2 hours. Wash 3 times for 10 minutes in cold PBS, place thymi in OCT, and freeze and store until use at -80&deg;C.</li>
	<li>For cryosectioning, spread OCT on a section 'block' and mount the embryo or dissected organs/tissues for sectioning.</li>
	<li>Cut frozen tissue into 10 &mu;m thick sections and collect on slides.</li>
	<li>Fix sections in acetone for 5-10 minutes. Wash 3 times in cold TBS.</li>
	<li>Block in 10% donkey serum/TBS in a humidity chamber at room temperature.</li>
	<li>Incubate sections for 1 hour-overnight with 100 &mu;l of primary antibodies in a humidity chamber at 4&deg;C: in this example, we use rat anti-mouse CD31 (1:100) to label endothelium, and goat anti-mouse PDGFR-&beta; (1:100) to label perivascular cells. It is useful to cover slides with individually cut Parafilm strips to ensure that the antibody is uniformly spread across the section.</li>
	<li>Following incubation with primary antibody, wash sections 3 times in cold TBS. Incubate with 100 &mu;l of appropriate secondary antibodies for 30 minutes minimum.</li>
	<li>Wash 3 times in cold TBS. Add 100 &mu;l of mounting media to each slide and a cover glass.</li>
	<li>Allow slides to dry in a dark storage area.</li>
	<li>Proceed to 'Image Acquisition' section.</li>
</ol>
4. Multi-color labeling of thymus vasculature and perivascular cells for vibratome sections (Continue from Section 1, Step 7)
<ol>
	<li>Dissect out thymus lobes from embryo and rinse in cold PBS.</li>
	<li>Fix thymus in 4% PFA/PBS at room temperature for 2 hours.</li>
	<li>Wash in PBS-Triton X (0.15%) 3 times, 10 minutes and place thymi in a small plastic cartridge and submerge in 4% low melt agarose/PBS (&tilde;4&deg;C). The thymus should be in contact with the bottom of the cartridge.</li>
	<li>Allow agarose to solidify on ice (3-5 minutes). Use a razor blade to cut off excess agarose. Add glue to the vibratome block and adhere sample to the block.</li>
	<li>Add cold PBS to vibratome water bath until the sample and blade are immersed.</li>
	<li>Set speed and amplitude (high amplitude and low-moderate speed is ideal for soft thymus sections). The amplitude should be reduced if sections break up due to excess agitation.</li>
	<li>Cut 50 um sections.</li>
	<li>Using a paintbrush, collect sections in a 24-well microplate in cold PBS.</li>
	<li>Block sections in 500 &mu;l of 10% donkey serum in PBS-Triton X (0.15%) for 30 minutes.</li>
	<li>Incubate sections for 8 hours to overnight with primary antibody, such as anti-CD31 and anti-PDGFR-&beta;, in a covered 24-well microplate.</li>
	<li>Wash 3 times in PBS-Triton X (0.15%) over a total of 8 hours at 4&deg;C.</li>
	<li>Block sections in 10% donkey serum in PBS-Triton X (0.15%) for 30 minutes.</li>
	<li>Incubate sections for 8 hours to overnight at 4&deg;C with appropriate secondary antibodies.</li>
	<li>Wash 3 times in PBS-Triton X (0.15%) over a total of 8 hours at 4&deg;C.</li>
	<li>Re-fix samples in 4% PFA/PBS for 30 minutes on ice.</li>
	<li>Wash 3 times in PBS-Triton X (0.15%) over 30 minutes on ice.</li>
	<li>Dehydrate samples through a graded MeOH/PBS-Triton X series: 25% MeOH, 50% MeOH, 75% MeOH, and 100% MeOH at 10 minutes for each step. Replace 100% MeOH with fresh MeOH after 10 minutes and incubate for 1 hour at room temperature.</li>
	<li>In a glass container, mix BABB (Benzyl Alcohol:Benzyl Benzoate) in a 1:2 ratio. Combine BABB with MeOH for a final concentration of 50% BABB and 50% MeOH. Incubate the sample in BABB:MeOH for 10-15 minutes.</li>
	<li>Transfer sample to a glass container with 100% BABB and incubate for 10-15 minutes or until cleared, at room temperature.</li>
	<li>Fill depression slide (0.7mm depth) with fresh 100% BABB and transfer sample to the side. Add cover glass (No. 1.5) and seal with 2-3 coats of nail polish. Allow nail polish to harden in the dark at room temperature, then store sample at 4&deg;C. 
	Note: Slides must be completely sealed prior to confocal image acquisition. Images should be acquired within 12-24 hours, as fluorescent dyes can fade in BABB.</li>
	<li>Proceed to 'Image Acquisition' section.</li>
</ol>
5. Image acquisition
<ol>
	<li>Image 10 &mu;m frozen sections with a confocal microscope using the Plan-Apochromat 20X/0.8 objective (512 X 512 Pixels) with 488- (FITC-dextran/GSL 1 - isolectin B4), 543-, and 633-nm laser lines.</li>
	<li>Acquire confocal z-sections of whole mount skin and 50 &mu;m agarose-embedded sections using the Plan-Apochromat 10X/0.4 objective (512 X 512 Pixels) with 488- (FITC-dextran/GSL 1 - isolectin B4), 543-, and 633-nm laser lines. Serial Z-sections should be collected sequentially at 1-micron for each respective channel.</li>
	<li>Reconstruct serial Z-sections using Zeiss Axiovision 4.6 or other image analysis software.</li>
</ol>
6. Representative Results:
Efficient labeling of the embryonic vasculature is critical for assessing blood vessel defects in embryonic mice. Figure 1 shows specific labeling of E16.5 thymus blood vessels (1A-B) and co-labeling with CD31 (1B), in addition to staining of the right and left ventricles (1E-F), respectively. The GSL I-isolectin B4 protocol for cryosections as described in sections 1, 3, and 5 was used in these experiments. Whole-mount labeling of the skin blood vessel network on E16.5 mice, using the protocols described in sections 1, 2, and 5 is shown in Figure 1C-D.
<img alt="Figure 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> 
Figure 1 Legend. FITC GSL I - isolectin B4 facial vein injections into E16.5 mouse embryos. a. Cryosection of embryonic thymus following injection. b. Merge of CD31 co-labeling with isolectin B4. c. and d. Whole-mount of embryonic skin vasculature following injection. e. and f. Cryosection of embryonic heart e. (right ventricle) f. (left ventricle) following injection.

<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>

No conflicts of interest declared.

Whole-mount and PECAM-1 (CD31) staining on sections are the conventional methods for labeling the vasculature in embryonic mice. These methods require the use of direct and/or indirect immunofluorescence, and detergents to permeabilize mouse tissue. This proves to be a rather timely process. Here, we have employed FITC-dextran or isolectin B4 facial vein injections to directly label the embryonic vasculature, thereby eliminating the requirement for antibody labeling steps. Furthermore, this method allows the a...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Sigma</td>
			<td>FD150S-1G</td>
		</tr>
		<tr>
			<td>Fluorescein labeled GSL 1 - isolectin B4	</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 (O.C.T.</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 Serum</td>
			<td>Jackson	</td>
			<td>017-000-121</td>
		</tr>
		<tr>
			<td>rat anti-mouse CD31,</td>
			<td>BD Pharmingen</td>
			<td>558736</td>
		</tr>
		<tr>
			<td>goat anti-mouse PDGFR-&beta;	</td>
			<td>R&amp;D Systems</td>
			<td>	AF1042</td>
		</tr>
		<tr>
			<td>donkey anti-rat CD31 Alexa 647 (Invitrogen) 		</td>
			<td>Biolegend</td>
			<td>102516</td>
		</tr>
		<tr>
			<td>donkey anti-goat 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>Benzyl Alcohol</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 slides</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>Cover Glass (22X22)-1.5</td>
			<td>Thermo Scientific</td>
			<td>152222</td>
		</tr>
		<tr>
			<td>Zeiss LSM 510 Meta Confocal Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps 		</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 and 24 well microplates</td>
			<td>Evergreen Scientific</td>
			<td>222-8044-01F</td>
		</tr>
		<tr>
			<td>Superfrost/Plus
Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
		</tr>
		<tr>
			<td>4mL clear vials		</td>
			<td>National Scientific</td>
			<td>B7800-2</td>
		</tr>
	</tbody>
</table>

a method for labeling vasculature in embryonic mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 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

This article describes a method for labeling embryonic skin and thymus blood vessels.

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

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Research

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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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ...