Research Article

En Face Preparation of Mouse Blood Vessels

DOI:

10.3791/55460

⸱

May 19th, 2017

 ,  ,  , 
1Department of Cardiology, Division of Internal Medicine, University of Texas MD Anderson Cancer Center, 2Department of Radiation Oncology, University of Texas MD Anderson Cancer Center

In This Article

Summary

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A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

Abstract

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Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

Introduction

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For histopathological studies by light microscopy, three dimensional pieces of biological tissues are routinely processed for paraffin embedment followed by sectioning and staining. A tissue sample that has been paraffin-embedded may be several millimeters in all three dimensions. However, for the purpose of light microscopy, it must be first sectioned so that light can pass through and then stained so that the thin section yields enough contrast for imaging. Because sectioned specimens are usually 5-10 µm in thickness, one sees only a very small fraction of the whole specimen in two dimensions at a time. It is possible to collect sequential sections and, after i....

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Protocol

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The protocols for the mouse partial carotid artery ligation and isolation of the mouse aorta and carotid artery for en face immunostaining are approved by the Institutional Animal Care and Use Committee (IBT 2014-9231).

1. Left Partial Carotid Artery Ligation

  1. Prepare the surgical space by placing a 12 inch x 14 inch heating pad on the table and cover the pad and table top with a large clean surgical drape. Adjust the arm of the boom stand so that the stereomicroscope's field of view is in the center area of the heating pad.
  2. Turn on the heating pad on the table and set the 3-setting control dial to the mediu....

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Results

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A typical en face immunofluorescence image of the endothelium is shown in Figure 3. This image shows a single optical section of a mouse aorta taken near the opening of an intercostal artery (the large dark egg-shaped area). The aorta was double-stained with anti-VE-cadherin (green) and anti-VCAM-1 (vascular cell adhesion molecule-1) (red). Each endothelial cell is outlined with a green linear staining at the adherens junction. Due to minor unevenness of the specimen, som.......

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Discussion

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When handling mouse blood vessels, it is important to remember that the endothelium is fragile and that any excessive mechanical force will damage endothelial cells. For example, endothelial cells break or detach from the vessel wall if the vessel is perfused too forcefully, which can easily happen when the vasculature is perfused using a hand-operated syringe.

To obtain constant perfusion pressure, we use a gravity perfusion system with a 120 cm water column pressure. It has been reported tha.......

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Acknowledgements

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The research activities of the authors are supported by grants from the National Institute of Health to Dr. Abe (HL-130193, HL-123346, HL-118462, HL-108551).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.9% Sodium Chloride injection solution USP 500 mL bagFisher ScientificNC9788429
12-well platesFisher Scientific12556005
6-0 coated vicryl sutureEthiconJ833G
AF488 goat anti-rat IgG Life TechnologiesA11006
AF546 goat anti-rabbit IgG Life TechnologiesA11035
Anti-CD144 (Ve-Cad)BD BiosciencesBD555289
Anti-VCAM-1 (H-276) Rabbit polyclonal IgGSanta Cruze BiotechnologySc-8304
Aoto Flow SystemBraintree ScientificEZ-AF9000
Autoclave Wrap. 24 inch x 24 inchCardinal Health4024
Blunt retractors, 2.5 mm wideFine Science Tools18200-10
Caprofen (Rimadyl) zoetisNADA#141-199
Chlorhexidine Scrub, 2%Med-Vet InternationalRXCHLOR2-PC
Curity gauze sponges 2 x 2Cardinal HealthKC2146
Electric heating pad, 12 x 14Fisher ScientificNC0667724
Extra Fine Graefe ForcepsFine Science Tools11152-10
Iris ScissorsFine Science Tools14090-11
Micro cover glass 22 mm x 50 mmVWR48393059
Microscope SlidesFisher Scientific12-550-18
normal goat serumEquitech-BioGS05
Paraformaldehyde Solution 4% in PBSSanta Cruze BiotechnologySC-281692
Petri dishes 100 mm x 15 mmFisher ScientificFB0875713
Prolong Gold Antifade mountant with DAPILife TechnologiesP-36935
Puritan cortton swabsVWR10806-005
Puritan Mini cotton tipped aplicatorsVWR82004-050
Round handled Needle HolderFine Science Tools12076-12
Silk Suture 6/0Fine Science Tools18020-60
Spring scissorsROBOZRS-5601
Strabismus ScissorsFine Science Tools14075-09
Super Grip ForcepsFine Science Tools00649-11
Transparent DressingCardinal HealthTD-26C
Triton X-1000Fisher ScientificAC327371000

References

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  1. Jelev, L., Surchev, L. A novel simple technique for en face endothelial observations using water-soluble media -'thinned-wall' preparations. J Anat. 212 (2), 192-197 (2008).
  2. Neill, J. F. The effect on venous endothelium of alterations in blood flow through the vessels in vein walls, and the possible relation to thrombosis. Ann Surg....

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Tags

Keywords En FaceMouse Blood VesselsAortaCarotid ArteryImmunostainingVascular CellsCarotid Artery LigationSurgical ProcedureAnesthetized MouseSalivary GlandsInternal Carotid ArteryExternal Carotid ArteryLigationOccipital ArterySuturesThoracic Cavity