1Department of Cellular Biology, University of Georgia, 2Centre for Immunology and Infection, Department of Biology and HYMS, University of York, 3Department of Genetics, University of Georgia
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Bryson, J. L., Coles, M. C., Manley, N. R. A Method for Labeling Vasculature in Embryonic Mice. J. Vis. Exp. (56), e3267, doi:10.3791/3267 (2011).
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-β 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.
1. Fluorescein labeled dextran and GSL I-isolectin B4 facial vein injections to label embryonic vasculature
2. Whole-mount analysis of skin vasculature
3. Multi-color labeling of thymus and heart vasculature and perivascular cells for cryosections (Continue from Section 1, Step 7)
4. Multi-color labeling of thymus vasculature and perivascular cells for vibratome sections (Continue from Section 1, Step 7)
5. Image acquisition
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.
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.
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 assessment of the functional status of the vasculature, as 'leaky' phenotypes will be revealed by this method but not by conventional antibody staining methods.
We have validated the efficiency of this technique by analyzing sections of embryonic thymus, and heart, as well as whole mounts of skin vasculature shortly after facial vein injections. Additionally, we have stained thymus sections for CD31 following FITC-dextran or isolectin B4 injections to demonstrate that FITC specifically labels embryonic vascular structures. Therefore FITC-dextran or isolectin B4 facial vein injections in embryos can be utilized to rapidly assess vascular phenotypes in multiple organs throughout development. In addition, this method allows researchers to determine the specific time during development in which the peripheral embryonic vasculature connects to an organ of interest, via angiogenesis. We have included an antibody labeling protocol that is compatible with this FITC-dextran or isolectin B4 protocol. Using this protocol, PDGFR-β, SMA-α, and other antibodies, which label structural components of blood vessels, may be employed to further assess the nature of embryonic vascular defects.
Important Notes: A dye such as Fast Green can be added to the FITC-dextran/GSL 1 - isolectin B4 solution in order to visualize the mixture as it is injected into the facial vein. Shortly after FITC-dextran injection, the dye can be observed in the umbilical vein and in blood vessels throughout the yolk sac and placenta. At this point, the umbilical cord should be removed from the embryo. In the event that dye is not detected in the allantoic stalk (umbilical artery and vein) the technique likely failed. We have observed that if the needle does not directly enter the facial vein, FITC-dextran/ Fluorescein labeled GSL 1 - isolectin B4 accumulates in adjacent areas of the face. We have also observed that FITC-dextran labels the entire vasculature, while GSL 1 - isolectin B4 labels most embryonic vascular structures, but also non-vascular cells in the liver. A fluorescence-dissecting microscope can be used to test whether the injection was successful.
C57Bl6/J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Embryos were generated via wild type, timed matings. Experiments were approved by the University of Georgia's Institutional Animal Care and Use Committee.
No conflicts of interest declared.
This work was supported by grant numbers R01AI055001 and R01AI082127 from NIAID to NRM and SREB Dissertation Fellowship Award to JLB.
|Fluorescein labeled GSL 1 - isolectin B4||Vector Laboratories||FL-1201|
|Fast Green||MP Biomedicals||195178|
|Fetal Bovine Serum||Atlanta Biologicals||S11550|
|Optimal Cutting Temperature Compound (O.C.T.||VWR international||25608-930|
|rat anti-mouse CD31,||BD Biosciences||558736|
|goat anti-mouse PDGFR-β||R&D Systems||AF1042|
|donkey anti-rat CD31 Alexa 647 (Invitrogen)||Biolegend||102516|
|donkey anti-goat Alexa 594 (Invitrogen)||Invitrogen||A11058|
|Triton X -100||Sigma-Aldrich||X-100|
|Low melt agarose/PBS||Sigma-Aldrich||A9414-25G|
|Benzyl Alcohol||Acros Organics||148390010|
|Benzyl Benzoate||Acros Organics||105860010|
|Depression slides||Fisher Scientific||S175201|
|Fluorogel||Electron Microscopy Sciences||17985-10|
|Cover Glass (22X22)-1.5||Thermo Fisher Scientific, Inc.||152222|
|Zeiss LSM 510 Meta Confocal Microscope||Carl Zeiss, Inc.|
|Micro dissecting forceps||Roboz Surgical Instruments Co.||RS-5135|
|Parafilm No. OM992||Fisher Scientific||13-374-16|
|12 and 24 well microplates||Evergreen Scientific||222-8044-01F|
|Superfrost/PlusMicroscope Slides||Fisher Scientific||12-550-15|
|4mL clear vials||National Scientific Company||B7800-2|