The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels
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The purpose of this study was to develop the methods needed to enable the application of Immunofluorescent Array Tomography (IAT), a novel three-dimensional (3D) microscopy technique, to murine blood vessels. The anterior and posterior regions of the infrarenal aorta of 8–10 week old C57BL6 mice were evaluated. Staining and image analysis methods were developed. Antibody selection, primary antibody concentration, co-staining with multiple primary antibodies, and the multi-cycle staining design were optimized to produce positive and specific staining of elastin (1:50 dilution), smooth muscle cell actin (SMCA, 20 μg mL−1), and collagen type I (10 μg mL−1). Non-specific interactions were limited by optimizing secondary antibody staining conditions (1:200 dilution, 30 min). SMCA and collagen type I were stained in the first cycle, elastin was stained in the second cycle, and nuclei were stained in both cycles. Algorithms were developed to quantify volume fractions of medial elastin, SMCA, and nuclei, as well as adventitial collagen type I. Elastin thickness, spacing between elastin lamellae, elastin fragmentation, media wall thickness, nuclei aspect ratio, and nuclei amount were also quantified. We have qualitatively and quantitatively characterized the 3D microstructure and cellular morphology of the anterior and posterior infrarenal murine aorta using IAT.
KeywordsImmunofluorescence microscopy Three-dimensional microstructure Murine blood vessels Infrarenal aorta Elastin Collagen type I Smooth muscle cells Volume fraction Cellular morphology
The authors gratefully acknowledge all members of the Smith Laboratory at Stanford University, especially Brad L. Busse, Dr. Kristina D. Micheva, Dr. Nancy A. O’Rourke, and Dr. Gordon X. Wang, for their technical expertise in IAT, support in developing the presented methods, and review of the manuscript. We also acknowledge Dr. Joan M. Greve for her encouragement, support, and critical review of the manuscript. The authors also gratefully acknowledge all staff members of the Cell Sciences Imaging Facility at Stanford University, especially Jon W. Mulholland and Ibanri Phanwar, for technical guidance and all array generation. We would also like to acknowledge Dr. Andrew J. Connolly in the Pathology Department at Stanford University for his expertise in vascular histology and staining. S. Saatchi was supported by a Stanford University Bio-X Graduate Student Fellowship sponsored by Amgen Inc. This research was funded in part by the NIH (1P50HL083800). The authors do not have any conflicts of interest.
- 3.Berry, C. L., T. Looker, and J. Germain. The growth and development of the rat aorta. I. Morphological aspects. J. Anat. 113(Pt 1):1–16, 1972.Google Scholar
- 6.Bunton, T. E., N. J. Biery, L. Myers, B. Gayraud, F. Ramirez, and H. C. Dietz. Phenotypic alteration of vascular smooth muscle cells precedes elastolysis in a mouse model of Marfan syndrome. Circ. Res. 88(1):37–43, 2001.Google Scholar
- 8.Clark, J. M., and S. Glagov. Transmural organization of the arterial media. The lamellar unit revisited. Arteriosclerosis 5(1):19–34, 1985.Google Scholar
- 9.Dai, D., Y. H. Ding, D. A. Lewis, and D. F. Kallmes. A proposed ordinal scale for grading histology in elastase-induced, saccular aneurysms. AJNR Am. J. Neuroradiol. 27(1):132–138, 2006.Google Scholar
- 10.Davies, J. Q., and S. Gordon. Isolation and culture of murine macrophages. Methods Mol. Biol. 290:91–103, 2005.Google Scholar
- 13.Lopez-Candales, A., D. R. Holmes, S. Liao, M. J. Scott, S. A. Wickline, and R. W. Thompson. Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms. Am. J. Pathol. 150(3):993–1007, 1997.Google Scholar
- 14.Micheva, K. D., and S. J Smith. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55(1):25–36, 2007.Google Scholar
- 16.O’Connell, M. K., S. Murthy, S. Phan, C. Xu, J. Buchanan, R. Spilker, R. L. Dalman, C. K. Zarins, W. Denk, and C. A. Taylor. The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging. Matrix Biol. 27(3):171–181, 2008.CrossRefGoogle Scholar
- 18.Vanhecke, D., S. Asano, Z. Kochovski, R. Fernandez-Busnadiego, N. Schrod, W. Baumeister, and V. Lucic. Cryo-electron tomography: methodology, developments and biological applications. J Microsc. [Epub ahead of print], 2010.Google Scholar
- 19.Wasano, K., and T. Yamamoto. Tridimensional architecture of elastic tissue in the rat aorta and femoral artery—a scanning electron microscope study. J. Electron. Microsc. (Tokyo) 32(1):33–44, 1983.Google Scholar
- 20.Wolinsky, H., and S. Glagov. A lamellar unit of aortic medial structure and function in mammals. Circ. Res. 20(1):99–111, 1967.Google Scholar